Photon Irradiation Prompts Autophagy in Anaplastic Thyroid Cancer

Photon Irradiation Prompts Autophagy in Anaplastic Thyroid Cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Photon Irradiation Prompts Autophagy in Anaplastic Thyroid Cancer Sabine Wächter, Franziska Knauff, Silvia Roth, Norman Krasser-Gercke, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5193679/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Aug, 2026 Read the published version in BMC Cancer → Version 1 posted 4 You are reading this latest preprint version Abstract Although promising results have been obtained for anaplastic thyroid cancer (ATC) therapy, this therapy still needs to be improved. In addition to the currently approved therapies, radiotherapy alone or combined with adjuvant immunotherapy could be beneficial for patients affected by ATC. The patients affected by this aggressive solid malignancy could benefit from the modulation of autophagy in cancer cells. This study focused on detecting autophagy players in ATCs and bursting autophagy process via photon irradiation to induce decay in irradiated ATCs. The transcript expression of autophagy genes was detected in tumor tissue resected from 19 patients and in C643 cells, four primary ATC cell lines and primary follicular thyroid cell line (Nthy-ori-3-1) photons irradiated with 4 or 6 Gy. The protein level of Beclin1 was detected by immunofluorescence in 10/19 patients. The levels of autophagy markers were detected by RT‒qPCR and western blotting in irradiated cells. Autophagy and maturation of autophagosome vesicles were monitored in C643 cells stably transfected with the GFP-RFP-LC3B plasmid. All patients included in the study exhibited significant overexpression of autophagy transcripts. Additionally, the Beclin1 protein was expressed in resected tumor tissue. Furthermore, the analysis of autophagy-related gene transcripts revealed significant increases in the expression of these genes in C643, Patient 2 and Patient 3 cells irradiated with 4 or 6 Gy. Additionally, irradiation with 4 or 6 Gy downregulated the expression of all the proteins involved in the autophagy process. Thus, the ongoing catabolic process was confirmed. Interestingly, the levels of AMPKα and its active phosphorylated form were strongly downregulated, which excluded its involvement in autophagy activation. 6 Gy photon irradiation caused an increase in both green and red fluorescence in C643-derived spheroids. An increase in fluorescence was detectable for up to 28 days. The spheroids gradually exhibited an increase in fluorescence, which probably caused the dismantling of their ultrastructure. This finding provides evidence of the bursting of autophagy and its ability to affect spheroid integrity. Photon irradiation exacerbates autophagy in anaplastic thyroid cancer cells and could represent a valid target for focused therapy against this aggressive malignancy. autophagy radiotherapy anaplastic thyroid cancer poor prognosis targeted therapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 BACKGROUND Although anaplastic thyroid cancer (ATC) accounts for only 1–2% of all thyroid cancers (TCs), it contributes to a large proportion of all patients who die from TC ( 1 , 2 ). The aggressive growth of this rare carcinoma is characterized by rapid lymphogenic and hematogenic metastasis with infiltration of surrounding structures; consequently, patients have a poor prognosis, with a median survival of only 3 to 5 months after initial diagnosis ( 3 , 4 ). In the last two decades, ATC therapy has been based on a variety of treatment options, including surgery, radiotherapy and chemotherapy. Photon radiotherapy is the standard treatment for ATC and is associated with prolonged overall survival at high doses (≥ 60 Gy). As a result, intensity-modulated radiotherapy (IMRT) has become increasingly important for the treatment of ATC, as it has been shown to provide high and precise radiation intensity to tumors while sparing surrounding healthy tissue from nonspecific side effects ( 5 , 6 , 1 , 7 , 8 ). However, despite targeted, individualized treatment options, including IMRT, the poor prognosis of ATC has not significantly improved in the last 10 years ( 9 ). In contrast, the development of targeted treatment options such as immune checkpoint inhibitors (ICIs) and multikinase inhibitors (mKIs) appears to improve survival in patients with advanced or primarily unresectable ATC, which has already been exhausted by canonical therapy options ( 10 ). A previous study by our working group showed that photon radiation led to a decrease in cell viability in ATC cells alone or in combination with the ICI atezolizumab ( 11 ). According to current knowledge, radiation induces DNA damage through double-strand breaks (DSBs) and activates the intrinsic and extrinsic apoptotic pathways ( 12 ). In contrast, we were unable to detect any induction of apoptosis in irradiated ATCs ( 11 ). Accordingly, other cellular processes must be involved in photon-induced cell death in ATC. Therefore, this study focused on investigating whether photon irradiation can induce autophagy as a substitute mechanism for ATC cell death. MATERIALS and METHODS Cell lines C643 human anaplastic thyroid carcinoma cells kindly donated by Prof. A. Zielke (Diakonie-Klinikum Stuttgart; Stuttgart, Germany) and the human thyroid follicular epithelial cell line Nthy-ori-3-1 (MERCK - Sigma‒Aldrich Chemie GmbH, Schnelldorf Germany) were grown in RPMI 1640 (Gibco® by Life TechnologiesTM, Carlsbad, USA) supplemented with 10% fetal bovine serum (Gibco) and 10 U/ml penicillin and 100 µg/ml streptomycin (Gibco). The cells were kept under standard conditions (37°C, 5% CO 2 ) and routinely tested for Mycoplasma contamination ( 13 , 11 ). Patient samples Snap-frozen and formalin-fixed paraffin-embedded (FFPE) tumor tissue was collected from 19 patients affected by ATC who underwent surgical resection at the University Hospital Marburg between 2003 and 2023. Preparation of patient-derived human tumor tissue (PDTT) Patient-derived human tumor tissue (PDTT) was isolated from four surgically operated patients affected by ATC as previously described ( 14 ). All PDTTs were obtained from patients who were never treated with neoadjuvant therapy. The tumor tissue was immediately collected in 50 ml falcon tube with sterile phosphate-buffered saline (PBS) without Ca 2+ or Mg + (L1825 Biochrom, Berlin, Germany). The tissue was washed 3 times with sterile PBS to remove any tissue debris or blood. Afterward, the tissue was cut into small pieces with a sterile scalpel (Feather, Osaka, Japan). The small pieces were rinsed through a cell strainer (352350 BD Labware, Franklin Lakes, NJ, USA) and washed with Roswell Park Memorial Institute 1640 (RPMI1640) medium (FG1215 Biochrom, Berlin, Germany). The cell suspension was centrifuged at 1,500 rpm for 8 min at room temperature. The pellet was suspended in complete growth medium RPMI 1640 (Biochrom) supplemented with 10% fetal bovine serum (FBS; Biochrom), 10 U/mL penicillin and 100 g/mL streptomycin (Biochrom). The suspension was pipetted into a cell culture 6-well plate (83.3920 Sarstedt, Nümbrecht, Germany). After 2 h, the cell adhesion was monitored under contrast light microscope. Fresh medium was added regularly every second day. The cells were then trypsinized and transferred to 25 cm 2 flasks and were grown in RPMI 1640 (Biochrom) supplemented with 10% fetal bovine serum (FBS; Biochrom), 10 U/mL penicillin and 100 g/mL streptomycin (Biochrom) under standard conditions (37°C, 5% CO2). All cells were routinely tested for Mycoplasma contamination ( 14 ). Immunofluorescence staining of paraffin-embedded tissue Two-micron sections of 4% formaldehyde-fixed paraffin-embedded tumor tissue were cut, rehydrated and deparaffinized. Antigen retrieval was performed in citrate buffer (pH = 6) in a microwave at 480 W for 10 minutes. Endogenous peroxidase activity was blocked with 3% H 2 O 2 for 10 minutes. The sections were permeabilized with 0.5% Triton X-100 (Carl Roth Gmbh & Co. KG) in PBS (Life Technologies) for 10 minutes. Unspecific binding was blocked through a 30-minute incubation in 10% immunized serum. The slides were then incubated with a 1 µg/ml primary antibody against Beclin1 (ab114071; Abcam, Cambridge UK) in 1% BSA-PBS-0.5% Tween 20 overnight at 4°C. The bound primary antibody was labeled with 2 µg/ml Alexa Fluor® 488 goat anti-mouse IgG (H + L) secondary antibody. Nuclei were stained with 1 µg/ml Hoechst 33342 (Sigma‒Aldrich) in 1% BSA-PBST. After 90 minutes of incubation with the secondary antibody and Hoechst, the tissue slides were processed with a Vector® TrueVIEW™ Autofluorescence Quenching Kit (VECTOR Laboratories, Burlingame, USA) and mounted with VECTASHIELD® Vibrance™ Antifade Mounting Medium (VECTOR Laboratories). LAS AF and LAS X software (Leica Microsystems, Wetzlar Germany) was used for the analysis of fluorescence images acquired with a wide-field fluorescence microscope (Leica DM 5500) ( 15 ). Irradiation The cells were irradiated with an XRad 320iX irradiation cabinet (Precision X-ray, Inc., Denver, USA) at 8 mA and 320 kV at a dose rate of 1.0 Gy/min. A filter with 0.5 mm Al/0.5 mm Cu was employed ( 11 ). Quantitative RT‒PCR Total RNA was isolated from the cells and 19 tumor tissue samples by using an RNeasy Mini Kit (74106, QIAGEN, Hilden Germany) according to the manufacturer`s protocol. cDNA was reverse transcribed by using an iScriptTM cDNA Synthesis Kit (170–8891; Bio-Rad, Hercules, USA) on a FlexCycler (Analytik Jena AG, Jena, Deutschland). The primers used for human BECN1 (QT00004221), UVRAG (QT00034328), MAP1LC3B (QT00055069), SQSTM1 (QT00095676), TFEB (QT00069951), PRKAA1_1 (QT00009436), PRKAA2_1 (QT00042077) and GAPDH (QT01192646) were mixed with the GoTaq® qPCR Master Mix (Promega, Madison, USA) on an RT‒qPCR thermocycler CFX96TM Real-Time System (Bio-Rad Laboratories, Hercules, California USA). The results were analyzed with a Bio-Rad CFX-Manager (Bio-Rad Laboratories) and normalized to the GAPDH mRNA content for each sample. The raw data were further processed with Rest2009 (relative expression software tool V.2.0.13, Qiagen) ( 13 ). Western blot analysis Whole-cell lysates were isolated in Jie´s Buffer (10 mM NaCl, 0.5% NonidetP40, 20 mM Tris-HCL (pH 7.4), 5 mM MgCl2, 1 mM PMSF, Complete Protease Inhibitor and Phosphatase Inhibitor (Roche, Basel Switzerland)). The proteins were separated through SDS‒PAGE (NP0342, Life Technologies, Carlsbad, California, USA) and transferred to 0.2 µm nitrocellulose membranes (#1704158, Trans-Blot Turbo Transfer Pack, Bio-Rad Laboratories, USA) by semidry blotting with a Trans-Blot® TurboTM Transfer System (Bio-Rad Laboratories). The membranes were further sliced according to the molecular weight of the proteins of interest, blocked in 4% BSA (23208; Thermo Fisher Scientific, Waltham, MA, USA) in TBS-Tween 20 (0.5%) and incubated with primary antibodies against Beclin1 (ab114071; Abcam), UVRAG (U7508. Sigma-Aldrich, St. Louis, USA), LC3B (ab51520, Abcam), SQSTM1 (ab96706, Abcam), AMPK-α (2532S, Cell Signaling Technology, Danvers, USA); phospho-AMPK-α (T172) (2525S; Cell Signaling Technology); and β-actin (A5441; Sigma‒Aldrich, St. Louis, USA). The bound primary antibodies were detected by secondary horseradish-labeled goat anti-rabbit (A0545, Sigma‒Aldrich) and goat anti-mouse (A9917, Sigma‒Aldrich) antibodies and SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, USA). The resulting bands were quantified by using Fusion image capture (VILBER LOURMAT Deutschland GmbH, Eberhardzell, Germany) and a Bio1D analysis system (VILBER LORUMAT Deutschland GmbH) ( 13 ). Stable Transfection C643 cells were stably transfected with an E. coli plasmid encoding RFP-GFP-MAP1LC3B (ptfLC3 was a gift from Tamotsu Yoshimori [Addgene plasmid #21074; http://n2t.net/addgene:21074 ]; RRID:Addgene_21074) ( 16 ) by incubation with 20 µg/ml plasmid in serum-free medium and FUGENE® HD Transfection Reagent (Promega). Fresh medium containing the selective agent G-418 (Roche Diagnostics Gmbh, Risch-Rotkreuz, Switzerland) was added 96h after transfection. After 1 week of G418-dependent selection, the transfected cells (fluorescent) were collected by scratching with a pipette under a fluorescence microscope. The scratched cells were plated on a new dish with fresh medium containing 20 µg/ml G-418 ( 15 , 17 ). Autophagy assay A total of 5,000 C643 cells stably transfected with RFP-GFP-MAP1LC3B were seeded in a round bottom low-attachment plate (Corning Spheroid Microplate 4515, Corning, USA) for 4 days. The transfected cells were firstly grown as spheroids and were then irradiated with 6 Gy. The green and red fluorescence intensity was continuously acquired by an IncuCyte® S3 Live-Cell Analysis System (Sartorius, Göttingen, Germany) and with a confocal microscope (Leica TCS SP8). By using a 488 nm laser (GFP) and 552 nm laser (RFP) with 5x objective One planar in the middle of the spheroids ( 15 ). Protein interaction Maps showing the protein interactions of the autophagy players were generated and downloaded from STRING v12.0. Statistical analysis Unless otherwise stated, all the experiments were performed in triplicate and repeated at least three times. The data were collected using Excel (Microsoft Office). Significance was calculated using the t test for paired samples. P < 0.05 was considered to indicate statistical significance (*). Ethical approval The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Ethics Committee of University Hospital of Marburg (No. 123/19). Informed consent was obtained from all subjects involved in the study. RESULTS Expression of autophagy players in ATC patient tissue In addition to the several autophagy genes that transcribe proteins involved in the autophagosome synthesis process and catabolic activity, this study focused on the detection of the BECN1 , MAP1LC3B , UVRAG and SQSTM1 genes, which are responsible for autophagosome vesicle nucleation, maturation and elongation, as previously described (13, 18). Additionally, the study focused on the detection of the transcription factor EB ( TFEB ), which is responsible for the transcription of autophagy genes, and the detection of PRKAA1_1 and PRKAA2_1 , the two genes responsible for the transcription of the two subunits of the kinase AMPKa (cyclic adenosine monophosphate kinase), which is a key element in linking metabolic signaling to autophagosome formation(18, 15). As shown in Figure 1A, the BECN1 transcript was detectable in all 19 patient samples included in the study, and its expression was more stable than that in human follicular thyroid cells. Furthermore, the expression levels of MAP1LC3B , UVRAG and SQSTM1 were significantly upregulated (median value 22.8-fold; 13.4-fold; 36.6-fold). Nonetheless, the TFEB transcript was also significantly overexpressed (median value 56.9-fold). The transcripts for AMPKa, PRKAA1_1 and PRKAA2_1 were both overexpressed (8.9- and 10.2-fold median values). However, PRKAA2_1 was detectable in only 9 tumor tissue samples. The protein Beclin1 was detected by immunofluorescence in 10 patient samples. Figure 1B highlights the overall expression of Beclin1 in eight out of the 10 patients affected by ATC. Lower magnification micrographs of all 10 patients are included in Suppl. Figure 1. Photon irradiation induces the overexpression of autophagy-related gene transcripts C643 cells, four primary tumor-derived cell lines (Patients 1-4) and human follicular epithelial thyroid cells (Nthy-ori-3-1) were photon irradiated with 4 or 6 Gy. Seven days after exposure, the expression of the TFEB , BECN1 , UVRAG , SQSTM1 , MAP1LC3B , PRKAA1_1 and PRKAA2_1 genes was detected in all the cells. Exposure to 4 Gy caused significant overexpression of TFEB and SQSTM1 in Nthy-ori-3-1, C643, Patient 2 and Patient 4 cells. Additionally, C643 cells were characterized by the significant overexpression of all the autophagy-related transcripts. However, Patient 1 exhibited stable expression of all the targets, and Patient 3 exhibited significant overexpression of only the TFEB , BECN1 and MAP1LC3B transcripts. Exposure to 6 Gy, similar to 4 Gy, caused significant overexpression of autophagy genes in all the cells. Notably, C643 cells were, once again, the most sensitive in terms of overexpressing the autophagy transcripts. Patient 1 cells once again exhibited stable expression of the autophagy transcripts after exposure to 6 Gy. Photon irradiation affects the protein level of autophagy players Based on the current findings showing the ability of irradiation to modulate autophagy at the transcriptional level, further experiments have focused on detecting the protein levels of autophagy factors in irradiated anaplastic thyroid cancer cells. As shown in Figure 3, exposure to 4 Gy caused the downregulation of Beclin, p62 and LC3B-I in all the anaplastic thyroid cancer cells. However, the protein level of LC3B-II was stable or slightly decreased (patient 3 cells). Nthy-ori-3-1 cells were the only cells that stably expressed all four proteins. Irradiation with 6 Gy also downregulated Beclin, p62, LC3B-I and LC3B-II in all cancer cells. Only Patient 2 cells were characterized by a stable protein level of LC3B-I and a significant increase in LC3B-II. The protein levels of the autophagy proteins were not altered after irradiation with 4 Gy in human follicular epithelial thyroid cells. Only 6 Gy irradiation caused a significant increase in Beclin and a significant decrease in p62 in the non-tumour cells. Modulation of AMPKa after irradiation of ATC cells cAMP kinase alpha is responsible for the phosphorylation of Unc-51 like autophagy activating kinase 1 (ULK1) and for signaling related to autophagosome formation. Recently, autophagy was shown to act independently of AMPKa in cancer(15, 13), and this kinase has been shown to inhibit autophagy (19) . Nonetheless, alterations in the expression of the downregulated gene PRKAA1 have recently been correlated with gastric and colorectal cancer risk and progression (20–23) . Here, it was observed that the ATC cells and the Nthy-ori-3-1 cells exposed to 4 and 6 Gy were characterized by the downregulation of AMPKa. In particular, all the cells isolated 7 days after irradiation exhibited significant downregulation of the protein level of AMPKa, with the exception of Patient 4 cells, which exhibited stable expression. Furthermore, the active phosphorylated form of AMPKa (P-AMPKa) was downregulated by irradiation at both 4 and 6 Gy in all the cells. Thus, irradiation was able to inhibit the activity of AMPKa not only by suppressing the total protein concentration but also by downregulating its active form. Live monitoring of the autophagy process in photon-irradiated ATCs C643 cells stably transfected with MAP1LC3B-GFP-RFP were seeded in a round bottom low-attachment plate for 4 days prior to exposure to 6 Gy photon irradiation. Immediately after exposure to 6 Gy photon irradiation, the C643 spheroid fluorescence and morphology were continuously tracked with IncuCyte for 28 days. Double-labeled LC3B allows the detection of autophagic maturation and terminal degradation activity after fusion with the lysosome. The terminal fusion of autophagosome vesicles and lysosomes causes the degradation of LC3B and acid-sensitive GFP . Instead, acid-stable RFP retains its fluorescence. As shown in Figure 5A, C643 cells exhibited basal autophagy at the time of exposure to 6 Gy photon irradiation, as indicated by consistent green and red fluorescence. The fluorescence intensity increased every day, and both fluorescence colors merged, as highlighted by the yellow‒brownish color after day 6 of exposure, resulting from the combination of green and red fluorescence. After 7 days of exposure to 6 Gy, the spheroid morphology started to dismantle, losing its three-dimensional structure and becoming untightened, while the green/red fluorescence remained stable (Suppl. Video). Similar results were observed via confocal microscopy (Figure 5B). In particular, these micrographs provided more detailed evidence of the dismantling of the spheroid morphology and the increase in fluorescence. This result supports previous findings highlighting the efficacy of irradiation in promoting autophagy, especially 7 days after exposure. DISCUSSION Anaplastic thyroid cancer is characterized by an extremely poor prognosis. Despite recent discoveries about the efficacy of personalized therapy based on mutation screening, patients affected by ATC still need therapy to significantly improve their survival rate. Combined therapy with lenvatinib, a tyrosine kinase inhibitor; pembrolizumab, a monoclonal antibody against PD-1; and the combination of dabrafenib, a BRAF inhibitor; and trametinib, a MAPK inhibitor, has shown promising effects on these patients (24). Unfortunately, prolonged treatment has led to relapse of the malignancy, thus affecting patient progression-free survival (25). For this reason, a second-line/adjuvant therapy is strongly needed to overcome the loss of efficacy of first-line therapy. The efficacy of radiotherapy in treating ATC has been highlighted previously (11, 26, 27). In particular, our previous study demonstrated that photon therapy is able to block the proliferation of ATC cells, thus preventing colony formation. The study could exclude the possibility of apoptosis as a cell death mechanism after irradiation. In fact, radiation can increase the protein level of PD-L1. Its inhibition mediated by the administration of atezolizumab exacerbates the inhibitory effect of radiotherapy. Thus, other cellular processes could be involved in the cellular decay prompted by photon irradiation. The current study focused on the ability of photon irradiation to promote autophagy as an alternative cell death mechanism. Autophagic cell death has been previously shown to be induced in anaplastic thyroid cancer cells by the administration of different compounds, thus revealing that this catabolic process is a promising therapeutic target for ATC (13). The combination of tyrosine kinase or pandeacetylase inhibitors with PD-L1 blockers, as well as berberine or even piperlongumine and capsaicin, has shown the ability to lead to autophagic cell death (28, 29, 13, 30). Here, it was shown for the first time that photon irradiation promoted autophagy in ATC cells. In particular, after seven days, 4 and 6 Gy irradiation caused the overexpression of the transcripts of the most related autophagy genes, TFEB, BECN1, UVRAG, SQSTM1 MAP1LC3B, PRKAA1_1 and PRKAA2_1 . Additionally, the protein levels of the autophagy players Beclin, LC3B-I, LC3B-II and p62 were significantly downregulated seven days after photon irradiation. The protein levels of AMPKa and its active phosphorylated form were also downregulated in irradiated cells. Thus, excluding definitively, its role in autophagy modulation. Similar effects of AMPKa have already been shown in pancreatic neuroendocrine neoplasia, where AKT exerts an inhibitory effect on AMPKa (15). However, these findings do not imply a negative modulation of autophagy that could be promoted at the transcriptional level by the cAMP responsive element or by the transcription factor TFEB (15). Moreover, after irradiation, autophagy continued to occur, and the process was further promoted, as shown by the increase in double fluorescence. This leads to a progressive dismantle of the ultrastructure of C643 cell-derived spheroids and ultimately cell decay. Anaplastic thyroid cancer tissue resected from patients showed significant overexpression of the autophagy genes MAP1LC3B, SQSTM1, UVRAG, TFEB, PRKAA1_1 and PRKAA2_1 as well as the protein Beclin1. Thus, ATC is characterized by stable active autophagy that can be promoted to induce cell death. This option has been previously shown for several solid cancers (31, 32, 15). Furthermore, autophagic cell death can promote cell death when prompted by small drugs and immune checkpoint inhibitors, not only in anaplastic thyroid cancer (13) but also in other solid malignancies (17, 33). Additionally, autophagy is likely involved in anaplastic lymphoma kinase (ALK)-associated cancers (34). Its modulation seems able to overcome the therapeutic resistance of several ALK-associated malignancies. CONCLUSIONS This study showed, for the first time, the ability of photon irradiation to sensitize anaplastic thyroid cancer cells to autophagy. Thus, cell death occurs. It is not known whether irradiation-mediated autophagy can modulate PD-L1 in ATCs, as previously shown (35). This ability could lead to a potential strategy for personalized therapy to more efficiently attack anaplastic thyroid cancer cells and inhibit their interaction with the tumor environment and immune cells. Abbreviations ATC Anaplastic Thyroid Cancer GFP Green fluorescent protein RFP Red fluorescent protein LC3B Light Chain 3B AMPKa 5'-AMP-activated protein kinase alpha IMRT Intensity-modulated radiotherapy DSBs Double Strand Breaks ICI Immune checkpoint inhibitor mKI multikinase Inhibitor RPMI Roswell Park Memorial Institute FFPE Formalin Fixed Paraffin Embedded PDTT Patient-Derived Tumor Tissue PBS Phosphate-buffered saline FBS Fetal bovine serum BSA Bovine Serum Albumin UVRAG UV Radiation Resistance-Associated Gene SQSTM1 Sequestosome 1 TFEB Transcription Factor EB PRKAA1_1 5'-AMP-activated protein kinase catalytic subunit alpha-1 PRKAA2_1 5'-AMP-activated protein kinase catalytic subunit alpha-2 GAPDH Glyceraldehyde 3-Phosphate Dehydrogenase MAP1LC3B Microtubule-associated protein 1A/1B light chain 3B BECN1 Beclin1 PD-1 Programmed cell death protein 1 PD-L1 Programmed death-ligand 1 Declarations Ethics approval and consent to participate The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Ethics Committee of University Hospital of Marburg (No. 123/19). Informed consent was obtained from all subjects involved in the study. Consent for publication Not applicable Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Competing interests None Funding This research was funded by an MIT Research Grant from Philipps University Marburg. Open access funding was provided by the Open Access Publishing Fund of Philipps University Marburg. Authors' contributions S.W. and D.K.B. performed the surgical resection and collected the material. F.K., S.R., N.K.-G., K.R. and P.D. performed the experiments. P.D. analyzed the data. S.W. and P.D. wrote the manuscript draft. D.K.B. revised the manuscript. Acknowledgement We are thankful to Stephanie Preising, Ulrike Theiss, Ekkehard Dikomey and the Institute of Radiation Biology and Molecular Radiation Oncology, Philipps University Marburg, for their technical support and use of the irradiation cabinet. References Bible KC, Kebebew E, Brierley J, Brito JP, Cabanillas ME, Clark TJ, et al. 2021 American Thyroid Association Guidelines for Management of Patients with Anaplastic Thyroid Cancer. Thyroid. 2021;31(3):337–86. Smallridge RC, Copland JA. Anaplastic thyroid carcinoma: pathogenesis and emerging therapies. Clin Oncol (R Coll Radiol). 2010;22(6):486–97. Haddad RI, Lydiatt WM, Ball DW, Busaidy NL, Byrd D, Callender G, et al. Anaplastic Thyroid Carcinoma, Version 2.2015. J Natl Compr Canc Netw. 2015;13(9):1140–50. Wächter S, Vorländer C, Schabram J, Mintziras I, Fülber I, Manoharan J, et al. Anaplastic thyroid carcinoma: changing trends of treatment strategies and associated overall survival. Eur Arch Otorhinolaryngol. 2020;277(5):1507–14. Pezzi TA, Mohamed ASR, Sheu T, Blanchard P, Sandulache VC, Lai SY, et al. Radiation therapy dose is associated with improved survival for unresected anaplastic thyroid carcinoma: Outcomes from the National Cancer Data Base. Cancer. 2017;123(9):1653–61. Fan D, Ma J, Bell AC, Groen AH, Olsen KS, Lok BH, et al. Outcomes of multimodal therapy in a large series of patients with anaplastic thyroid cancer. Cancer. 2020;126(2):444–52. Kwon J, Kim BH, Jung H-W, Besic N, Sugitani I, Wu H-G. The prognostic impacts of postoperative radiotherapy in the patients with resected anaplastic thyroid carcinoma: A systematic review and meta-analysis. Eur J Cancer. 2016;59:34–45. Haymart MR, Banerjee M, Yin H, Worden F, Griggs JJ. Marginal treatment benefit in anaplastic thyroid cancer. Cancer. 2013;119(17):3133–9. Lin B, Ma H, Ma M, Zhang Z, Sun Z, Hsieh I-Y, et al. The incidence and survival analysis for anaplastic thyroid cancer: a SEER database analysis. Am J Transl Res. 2019;11(9):5888–96. Maniakas A, Dadu R, Busaidy NL, Wang JR, Ferrarotto R, Lu C, et al. Evaluation of Overall Survival in Patients With Anaplastic Thyroid Carcinoma, 2000–2019. JAMA Oncol. 2020;6(9):1397–404. Wächter S, Roth S, Gercke N, Schötz U, Dikomey E, Engenhart-Cabillic R et al. Anti-Proliferative Effect of Radiotherapy and Implication of Immunotherapy in Anaplastic Thyroid Cancer Cells. Life (Basel) 2023; 13(6). Maier P, Hartmann L, Wenz F, Herskind C. Cellular Pathways in Response to Ionizing Radiation and Their Targetability for Tumor Radiosensitization. Int J Mol Sci 2016; 17(1). Wächter S, Knauff F, Roth S, Keber C, Holzer K, Manoharan J, et al. Synergic Induction of Autophagic Cell Death in Anaplastic Thyroid Carcinoma. Cancer Invest. 2023;41(4):405–21. Wächter S, Wunderlich A, Roth S, Mintziras I, Maurer E, Hoffmann S et al. Individualised Multimodal Treatment Strategies for Anaplastic and Poorly Differentiated Thyroid Cancer. J Clin Med 2018; 7(5). Matrood S, de Prisco N, Wissniowski TT, Wiese D, Jabari S, Griesmann H, et al. Modulation of Pancreatic Neuroendocrine Neoplastic Cell Fate by Autophagy-Mediated Death. Neuroendocrinology. 2021;111(10):965–85. Kimura S, Noda T, Yoshimori T. Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy. 2007;3(5):452–60. Di Fazio P, Waldegger P, Jabari S, Lingelbach S, Montalbano R, Ocker M, et al. Autophagy-related cell death by pan-histone deacetylase inhibition in liver cancer. Oncotarget. 2016;7(20):28998–9010. Di Fazio P, Matrood S. Targeting autophagy in liver cancer. Transl Gastroenterol Hepatol. 2018;3:39. Park J-M, Kim D-H. A paradigm shift: AMPK negatively regulates ULK1 activity. Autophagy 2023:1–3. Lee SJ, Kang BW, Chae YS, Kim HJ, Park SY, Park JS, et al. Genetic variations in STK11, PRKAA1, and TSC1 associated with prognosis for patients with colorectal cancer. Ann Surg Oncol. 2014;21(Suppl 4):S634–9. Mocellin S, Verdi D, Pooley KA, Nitti D. Genetic variation and gastric cancer risk: a field synopsis and meta-analysis. Gut. 2015;64(8):1209–19. Chen M, Jiang B, He B, Tang M, Wang P, Chen L, et al. Genetic variations in PRKAA1 predict the risk and progression of gastric Cancer. BMC Cancer. 2018;18(1):923. Yu S, Tu R, Chen Z, Song J, Li P, Hu F, et al. Association of PTGER4 and PRKAA1 genetic polymorphisms with gastric cancer. BMC Med Genomics. 2023;16(1):209. Cabanillas ME, Ryder M, Jimenez C. Targeted Therapy for Advanced Thyroid Cancer: Kinase Inhibitors and Beyond. Endocr Rev. 2019;40(6):1573–604. Zhang L, Feng Q, Wang J, Tan Z, Li Q, Ge M. Molecular basis and targeted therapy in thyroid cancer: Progress and opportunities. Biochim Biophys Acta Rev Cancer. 2023;1878(4):188928. Zhou W, Yue Y, Zhang X. Radiotherapy Plus Chemotherapy Leads to Prolonged Survival in Patients With Anaplastic Thyroid Cancer Compared With Radiotherapy Alone Regardless of Surgical Resection and Distant Metastasis: A Retrospective Population Study. Front Endocrinol (Lausanne). 2021;12:748023. de Leo S, Trevisan M, Fugazzola L. Recent advances in the management of anaplastic thyroid cancer. Thyroid Res. 2020;13(1):17. Shi X-Z, Zhao S, Wang Y, Wang M-Y, Su S-W, Wu Y-Z, et al. Antitumor Activity of Berberine by Activating Autophagy and Apoptosis in CAL-62 and BHT-101 Anaplastic Thyroid Carcinoma Cell Lines. Drug Des Devel Ther. 2023;17:1889–906. Lin T-H, Kuo C-H, Zhang Y-S, Chen P-T, Chen S-H, Li Y-Z et al. Piperlongumine Induces Cellular Apoptosis and Autophagy via the ROS/Akt Signaling Pathway in Human Follicular Thyroid Cancer Cells. Int J Mol Sci 2023; 24(9). Wu L, Xu S, Cheng X, Zhang L, Wang Y, Wu J, et al. Capsaicin inhibits the stemness of anaplastic thyroid carcinoma cells by triggering autophagy-lysosome mediated OCT4A degradation. Phytother Res. 2022;36(2):938–50. Matrood S, Melms LE, Bartsch DK, Di Fazio P. The Expression of Autophagy-Associated Genes Represents a Valid Footprint for Aggressive Pancreatic Neuroendocrine Neoplasms. Int J Mol Sci 2023; 24(4). Di Fazio P, Rusche FD, Roth S, Pehl A, Wächter S, Mintziras I, et al. Long Non-Coding RNA H19 Expression Correlates with Autophagy Process in Adrenocortical Carcinoma. Cancer Invest. 2022;40(3):254–67. Wirries A, Jabari S, Jansen EP, Roth S, Figueroa-Juárez E, Wissniowski TT, et al. Panobinostat mediated cell death: a novel therapeutic approach for osteosarcoma. Oncotarget. 2018;9(68):32997–3010. Frentzel J, Sorrentino D, Giuriato S. Targeting Autophagy in ALK-Associated Cancers. Cancers (Basel) 2017; 9(12). García-Pérez BE, Pérez-Torres C, Baltierra-Uribe SL, Castillo-Cruz J, Castrejón-Jiménez NS. Autophagy as a Target for Non-Immune Intrinsic Functions of Programmed Cell Death-Ligand 1 in Cancer. Int J Mol Sci 2023; 24(19). Additional Declarations No competing interests reported. Supplementary Files SupplFigure1.tif uncroppedblots1.tif uncroppedblots2.tif Cite Share Download PDF Status: Published Journal Publication published 25 Aug, 2026 Read the published version in BMC Cancer → Version 1 posted Editorial decision: Revision requested 03 Oct, 2024 Editor assigned by journal 03 Oct, 2024 Submission checks completed at journal 03 Oct, 2024 First submitted to journal 02 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5193679","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":362093169,"identity":"73ef7764-bddb-472c-9178-37db14b0166f","order_by":0,"name":"Sabine Wächter","email":"","orcid":"","institution":"Philipps University Marburg","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sabine","middleName":"","lastName":"Wächter","suffix":""},{"id":362093170,"identity":"6a4c5499-3143-43dd-916c-c31b75355922","order_by":1,"name":"Franziska Knauff","email":"","orcid":"","institution":"Philipps University Marburg","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Franziska","middleName":"","lastName":"Knauff","suffix":""},{"id":362093171,"identity":"571a5299-8d22-4cf1-a37e-ad00fcf8b1e2","order_by":2,"name":"Silvia Roth","email":"","orcid":"","institution":"Philipps University Marburg","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Silvia","middleName":"","lastName":"Roth","suffix":""},{"id":362093172,"identity":"426ec1d2-4de2-4484-866f-d39b957cdd03","order_by":3,"name":"Norman Krasser-Gercke","email":"","orcid":"","institution":"Philipps University Marburg","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Norman","middleName":"","lastName":"Krasser-Gercke","suffix":""},{"id":362093173,"identity":"ecb62ada-d348-4df4-b2cd-fda4e707a4a5","order_by":4,"name":"Katrin Roth","email":"","orcid":"","institution":"Philipps University Marburg","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Katrin","middleName":"","lastName":"Roth","suffix":""},{"id":362093174,"identity":"d370bf6d-7abf-4163-adbe-1c31379e0a5f","order_by":5,"name":"Detlef Klaus Bartsch","email":"","orcid":"","institution":"Philipps University Marburg","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Detlef","middleName":"Klaus","lastName":"Bartsch","suffix":""},{"id":362093175,"identity":"e2799d1b-85db-46cd-8d43-99f1eed716e3","order_by":6,"name":"Pietro Di Fazio","email":"data:image/png;base64,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","orcid":"","institution":"Philipps University Marburg","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Pietro","middleName":"Di","lastName":"Fazio","suffix":""}],"badges":[],"createdAt":"2024-10-02 15:08:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5193679/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5193679/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12885-026-16762-0","type":"published","date":"2026-08-25T15:57:39+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":68538838,"identity":"d5eb3163-49ec-4436-83be-d7fb8ac2e468","added_by":"auto","created_at":"2024-11-08 10:33:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2711669,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetection of autophagy markers in anaplastic thyroid cancer tissue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Box and whisker plots of \u003cem\u003eBECN1\u003c/em\u003e, \u003cem\u003eMAP1LC3B\u003c/em\u003e, \u003cem\u003eSQSTM1\u003c/em\u003e, \u003cem\u003eUVRAG\u003c/em\u003e, \u003cem\u003eTFEB\u003c/em\u003e, \u003cem\u003ePRKAA1_1\u003c/em\u003e and \u003cem\u003ePRKAA2_1 \u003c/em\u003etranscript levels. RNA was isolated from human ATC tissue. The tumor tissue expression was normalized to that of human follicular epithelial thyroid cells. The expression of the autophagy transcripts was normalized to that of GAPDH. The log10 means ±SEMs of triplicate samples are shown. (B) Immunofluorescence detection of Beclin in anaplastic thyroid cancer tissue. Tissue slices (10 µm) were deparaffinized and stained with a primary antibody against Beclin. The secondary antibody conjugated with Alexa Fluor 488 was used to identify the target protein by visualizing the green fluorescent spots. Nuclei were stained with Hoechst 33342 (blue). The magnification is 20×.\u003c/p\u003e","description":"","filename":"Figure1.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-5193679/v1/ce82b6284ec10c052b397381.png"},{"id":68538377,"identity":"79edf838-e338-4fe5-a53f-c2ad640cb7a8","added_by":"auto","created_at":"2024-11-08 10:25:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1018441,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of autophagy transcripts in irradiated anaplastic thyroid cancer cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTranscript levels of \u003cem\u003eTFEB\u003c/em\u003e, \u003cem\u003eBECN1\u003c/em\u003e, \u003cem\u003eUVRAG\u003c/em\u003e, \u003cem\u003eSQSTM1\u003c/em\u003e, \u003cem\u003eMAP1LC3B\u003c/em\u003e, \u003cem\u003ePRKAA1_1\u003c/em\u003e and \u003cem\u003ePRKAA2_1\u003c/em\u003e in Nthy-ori-3-1, C643, Patient 1, Patient 2, Patient 3 and Patient 4 cells 7 days after photon irradiation with 4 or 6 Gy. The irradiated cells were normalized to the untreated cells. GAPDH was detected as a housekeeping gene. *p\u0026lt;0.05 was regarded as significant for untreated vs. irradiated cells. The log10 means ±SEMs of triplicate samples are shown.\u003c/p\u003e","description":"","filename":"Figure2.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-5193679/v1/b32f7c86d9aeb1687d2ddae3.png"},{"id":68538837,"identity":"89c7f3ed-e46d-478a-9559-90ed426fcd23","added_by":"auto","created_at":"2024-11-08 10:33:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1487052,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetection of autophagy proteins in photon-irradiated ATCs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWestern blot membranes (upper) and densitometry (lower) of Beclin, LC3B-I, LC3B-II and p62 proteins from Nthy-ori-3-1, C643, Patient 1, Patient 2, Patient 3 and Patient 4 cells 7 days after photon irradiation at 4 and 6 Gy. Densitometry results were normalized to the beta-actin content. The mean densitometry values are presented in triplicate ± SEM. *p \u0026lt; 0.05 was considered to indicate statistical significance: untreated vs 4 or 6 Gy-irradiated cells. The map of the interactions between autophagy proteins was constructed with STRING v12.0.\u003c/p\u003e","description":"","filename":"Figure3.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-5193679/v1/795bafaaca8153b8a172c599.png"},{"id":68538379,"identity":"a7e16508-3b0d-4e4b-854d-4be3a97ed529","added_by":"auto","created_at":"2024-11-08 10:25:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1557310,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetection of the AMPKa protein in photon-irradiated ATCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWestern blot membranes (upper) and densitometry (lower) of AMPKa and its active phosphorylated form P-AMPKa in Nthy-ori-3-1, C643, Patient 1, Patient 2, Patient 3 and Patient 4 cells 7 days after photon irradiation with 4 or 6 Gy. Densitometry results were normalized to the beta-actin content. The mean densitometry values are presented in triplicate ± SEM. *p \u0026lt; 0.05 was considered to indicate statistical significance: untreated vs 4 or 6 Gy-irradiated cells. A map of the PRKAA interaction was constructed with STRING v12.0.\u003c/p\u003e","description":"","filename":"Figure4.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-5193679/v1/84d546cb24abd810ce821fd0.png"},{"id":68539961,"identity":"80967624-ad44-4f95-ba4d-765b09baba91","added_by":"auto","created_at":"2024-11-08 10:41:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4853925,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFluorescence-based monitoring of autophagy in photon-irradiated ATCs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStably transfected C643 cells were seeded in a Corning spheroid microplate. The cells were irradiated with 6 Gy. The green/red fluorescence was monitored for 28 days. The micrographs show fluorescence up to 14 days. (Left) Incucyte micrographs; the scale bar represents 400 µm. The magnification is 10×. (Right) Confocal microscopy micrographs; scale bar=300 µm; magnification=5x.\u003c/p\u003e","description":"","filename":"Figure5.tif.png","url":"https://assets-eu.researchsquare.com/files/rs-5193679/v1/8daa49a29b1d8da0f9ebc802.png"},{"id":118823659,"identity":"990ccaf3-e7ba-4bd1-8d0d-bb1eaa65886f","added_by":"auto","created_at":"2026-08-31 16:07:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10409788,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5193679/v1/0aca0984-9d86-4e98-977e-62493475ad2c.pdf"},{"id":68538384,"identity":"60ef1423-f7a1-4283-9c76-0cfd2c0cad6d","added_by":"auto","created_at":"2024-11-08 10:25:04","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":3421440,"visible":true,"origin":"","legend":"","description":"","filename":"SupplFigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-5193679/v1/348e93a0208732f829fafa61.tif"},{"id":68538381,"identity":"5f337588-cf6f-4918-b76c-d5b58192c631","added_by":"auto","created_at":"2024-11-08 10:25:04","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":2836468,"visible":true,"origin":"","legend":"","description":"","filename":"uncroppedblots1.tif","url":"https://assets-eu.researchsquare.com/files/rs-5193679/v1/377f7fe191ec25924d41e136.tif"},{"id":68538839,"identity":"d2fa469b-aab3-44b7-8f5f-c2b189f8a36a","added_by":"auto","created_at":"2024-11-08 10:33:04","extension":"tif","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":2559916,"visible":true,"origin":"","legend":"","description":"","filename":"uncroppedblots2.tif","url":"https://assets-eu.researchsquare.com/files/rs-5193679/v1/6b00a249019766bd6dcb90b2.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Photon Irradiation Prompts Autophagy in Anaplastic Thyroid Cancer","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eAlthough anaplastic thyroid cancer (ATC) accounts for only 1\u0026ndash;2% of all thyroid cancers (TCs), it contributes to a large proportion of all patients who die from TC (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The aggressive growth of this rare carcinoma is characterized by rapid lymphogenic and hematogenic metastasis with infiltration of surrounding structures; consequently, patients have a poor prognosis, with a median survival of only 3 to 5 months after initial diagnosis (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the last two decades, ATC therapy has been based on a variety of treatment options, including surgery, radiotherapy and chemotherapy. Photon radiotherapy is the standard treatment for ATC and is associated with prolonged overall survival at high doses (\u0026ge;\u0026thinsp;60 Gy). As a result, intensity-modulated radiotherapy (IMRT) has become increasingly important for the treatment of ATC, as it has been shown to provide high and precise radiation intensity to tumors while sparing surrounding healthy tissue from nonspecific side effects (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHowever, despite targeted, individualized treatment options, including IMRT, the poor prognosis of ATC has not significantly improved in the last 10 years (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn contrast, the development of targeted treatment options such as immune checkpoint inhibitors (ICIs) and multikinase inhibitors (mKIs) appears to improve survival in patients with advanced or primarily unresectable ATC, which has already been exhausted by canonical therapy options (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA previous study by our working group showed that photon radiation led to a decrease in cell viability in ATC cells alone or in combination with the ICI atezolizumab (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). According to current knowledge, radiation induces DNA damage through double-strand breaks (DSBs) and activates the intrinsic and extrinsic apoptotic pathways (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn contrast, we were unable to detect any induction of apoptosis in irradiated ATCs (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Accordingly, other cellular processes must be involved in photon-induced cell death in ATC.\u003c/p\u003e \u003cp\u003eTherefore, this study focused on investigating whether photon irradiation can induce autophagy as a substitute mechanism for ATC cell death.\u003c/p\u003e"},{"header":"MATERIALS and METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell lines\u003c/h2\u003e \u003cp\u003eC643 human anaplastic thyroid carcinoma cells kindly donated by Prof. A. Zielke (Diakonie-Klinikum Stuttgart; Stuttgart, Germany) and the human thyroid follicular epithelial cell line Nthy-ori-3-1 (MERCK - Sigma‒Aldrich Chemie GmbH, Schnelldorf Germany) were grown in RPMI 1640 (Gibco\u0026reg; by Life TechnologiesTM, Carlsbad, USA) supplemented with 10% fetal bovine serum (Gibco) and 10 U/ml penicillin and 100 \u0026micro;g/ml streptomycin (Gibco). The cells were kept under standard conditions (37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e) and routinely tested for Mycoplasma contamination (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePatient samples\u003c/h3\u003e\n\u003cp\u003eSnap-frozen and formalin-fixed paraffin-embedded (FFPE) tumor tissue was collected from 19 patients affected by ATC who underwent surgical resection at the University Hospital Marburg between 2003 and 2023.\u003c/p\u003e\n\u003ch3\u003ePreparation of patient-derived human tumor tissue (PDTT)\u003c/h3\u003e\n\u003cp\u003ePatient-derived human tumor tissue (PDTT) was isolated from four surgically operated patients affected by ATC as previously described (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). All PDTTs were obtained from patients who were never treated with neoadjuvant therapy. The tumor tissue was immediately collected in 50 ml falcon tube with sterile phosphate-buffered saline (PBS) without Ca\u003csup\u003e2+\u003c/sup\u003e or Mg\u003csup\u003e+\u003c/sup\u003e (L1825 Biochrom, Berlin, Germany). The tissue was washed 3 times with sterile PBS to remove any tissue debris or blood. Afterward, the tissue was cut into small pieces with a sterile scalpel (Feather, Osaka, Japan). The small pieces were rinsed through a cell strainer (352350 BD Labware, Franklin Lakes, NJ, USA) and washed with Roswell Park Memorial Institute 1640 (RPMI1640) medium (FG1215 Biochrom, Berlin, Germany). The cell suspension was centrifuged at 1,500 rpm for 8 min at room temperature. The pellet was suspended in complete growth medium RPMI 1640 (Biochrom) supplemented with 10% fetal bovine serum (FBS; Biochrom), 10 U/mL penicillin and 100 g/mL streptomycin (Biochrom). The suspension was pipetted into a cell culture 6-well plate (83.3920 Sarstedt, N\u0026uuml;mbrecht, Germany). After 2 h, the cell adhesion was monitored under contrast light microscope. Fresh medium was added regularly every second day. The cells were then trypsinized and transferred to 25 cm\u003csup\u003e2\u003c/sup\u003e flasks and were grown in RPMI 1640 (Biochrom) supplemented with 10% fetal bovine serum (FBS; Biochrom), 10 U/mL penicillin and 100 g/mL streptomycin (Biochrom) under standard conditions (37\u0026deg;C, 5% CO2). All cells were routinely tested for Mycoplasma contamination (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence staining of paraffin-embedded tissue\u003c/h3\u003e\n\u003cp\u003eTwo-micron sections of 4% formaldehyde-fixed paraffin-embedded tumor tissue were cut, rehydrated and deparaffinized. Antigen retrieval was performed in citrate buffer (pH\u0026thinsp;=\u0026thinsp;6) in a microwave at 480 W for 10 minutes. Endogenous peroxidase activity was blocked with 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 10 minutes. The sections were permeabilized with 0.5% Triton X-100 (Carl Roth Gmbh \u0026amp; Co. KG) in PBS (Life Technologies) for 10 minutes. Unspecific binding was blocked through a 30-minute incubation in 10% immunized serum. The slides were then incubated with a 1 \u0026micro;g/ml primary antibody against Beclin1 (ab114071; Abcam, Cambridge UK) in 1% BSA-PBS-0.5% Tween 20 overnight at 4\u0026deg;C. The bound primary antibody was labeled with 2 \u0026micro;g/ml Alexa Fluor\u0026reg; 488 goat anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L) secondary antibody. Nuclei were stained with 1 \u0026micro;g/ml Hoechst 33342 (Sigma‒Aldrich) in 1% BSA-PBST. After 90 minutes of incubation with the secondary antibody and Hoechst, the tissue slides were processed with a Vector\u0026reg; TrueVIEW\u0026trade; Autofluorescence Quenching Kit (VECTOR Laboratories, Burlingame, USA) and mounted with VECTASHIELD\u0026reg; Vibrance\u0026trade; Antifade Mounting Medium (VECTOR Laboratories). LAS AF and LAS X software (Leica Microsystems, Wetzlar Germany) was used for the analysis of fluorescence images acquired with a wide-field fluorescence microscope (Leica DM 5500) (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eIrradiation\u003c/h3\u003e\n\u003cp\u003eThe cells were irradiated with an XRad 320iX irradiation cabinet (Precision X-ray, Inc., Denver, USA) at 8 mA and 320 kV at a dose rate of 1.0 Gy/min. A filter with 0.5 mm Al/0.5 mm Cu was employed (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative RT‒PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated from the cells and 19 tumor tissue samples by using an RNeasy Mini Kit (74106, QIAGEN, Hilden Germany) according to the manufacturer`s protocol. cDNA was reverse transcribed by using an iScriptTM cDNA Synthesis Kit (170\u0026ndash;8891; Bio-Rad, Hercules, USA) on a FlexCycler (Analytik Jena AG, Jena, Deutschland). The primers used for human \u003cem\u003eBECN1\u003c/em\u003e (QT00004221), \u003cem\u003eUVRAG\u003c/em\u003e (QT00034328), \u003cem\u003eMAP1LC3B\u003c/em\u003e (QT00055069), \u003cem\u003eSQSTM1\u003c/em\u003e (QT00095676), \u003cem\u003eTFEB\u003c/em\u003e (QT00069951), \u003cem\u003ePRKAA1_1\u003c/em\u003e (QT00009436), \u003cem\u003ePRKAA2_1\u003c/em\u003e (QT00042077) and \u003cem\u003eGAPDH\u003c/em\u003e (QT01192646) were mixed with the GoTaq\u0026reg; qPCR Master Mix (Promega, Madison, USA) on an RT‒qPCR thermocycler CFX96TM Real-Time System (Bio-Rad Laboratories, Hercules, California USA). The results were analyzed with a Bio-Rad CFX-Manager (Bio-Rad Laboratories) and normalized to the GAPDH mRNA content for each sample. The raw data were further processed with Rest2009 (relative expression software tool V.2.0.13, Qiagen) (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWestern blot analysis\u003c/h3\u003e\n\u003cp\u003eWhole-cell lysates were isolated in Jie\u0026acute;s Buffer (10 mM NaCl, 0.5% NonidetP40, 20 mM Tris-HCL (pH 7.4), 5 mM MgCl2, 1 mM PMSF, Complete Protease Inhibitor and Phosphatase Inhibitor (Roche, Basel Switzerland)). The proteins were separated through SDS‒PAGE (NP0342, Life Technologies, Carlsbad, California, USA) and transferred to 0.2 \u0026micro;m nitrocellulose membranes (#1704158, Trans-Blot Turbo Transfer Pack, Bio-Rad Laboratories, USA) by semidry blotting with a Trans-Blot\u0026reg; TurboTM Transfer System (Bio-Rad Laboratories). The membranes were further sliced according to the molecular weight of the proteins of interest, blocked in 4% BSA (23208; Thermo Fisher Scientific, Waltham, MA, USA) in TBS-Tween 20 (0.5%) and incubated with primary antibodies against Beclin1 (ab114071; Abcam), UVRAG (U7508. Sigma-Aldrich, St. Louis, USA), LC3B (ab51520, Abcam), SQSTM1 (ab96706, Abcam), AMPK-α (2532S, Cell Signaling Technology, Danvers, USA); phospho-AMPK-α (T172) (2525S; Cell Signaling Technology); and β-actin (A5441; Sigma‒Aldrich, St. Louis, USA). The bound primary antibodies were detected by secondary horseradish-labeled goat anti-rabbit (A0545, Sigma‒Aldrich) and goat anti-mouse (A9917, Sigma‒Aldrich) antibodies and SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, USA). The resulting bands were quantified by using Fusion image capture (VILBER LOURMAT Deutschland GmbH, Eberhardzell, Germany) and a Bio1D analysis system (VILBER LORUMAT Deutschland GmbH) (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eStable Transfection\u003c/h3\u003e\n\u003cp\u003eC643 cells were stably transfected with an \u003cem\u003eE. coli\u003c/em\u003e plasmid encoding RFP-GFP-MAP1LC3B (ptfLC3 was a gift from Tamotsu Yoshimori [Addgene plasmid #21074; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://n2t.net/addgene:21074\u003c/span\u003e\u003cspan address=\"http://n2t.net/addgene:21074\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e]; RRID:Addgene_21074) (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) by incubation with 20 \u0026micro;g/ml plasmid in serum-free medium and FUGENE\u0026reg; HD Transfection Reagent (Promega). Fresh medium containing the selective agent G-418 (Roche Diagnostics Gmbh, Risch-Rotkreuz, Switzerland) was added 96h after transfection. After 1 week of G418-dependent selection, the transfected cells (fluorescent) were collected by scratching with a pipette under a fluorescence microscope. The scratched cells were plated on a new dish with fresh medium containing 20 \u0026micro;g/ml G-418 (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAutophagy assay\u003c/h2\u003e \u003cp\u003eA total of 5,000 C643 cells stably transfected with RFP-GFP-MAP1LC3B were seeded in a round bottom low-attachment plate (Corning Spheroid Microplate 4515, Corning, USA) for 4 days. The transfected cells were firstly grown as spheroids and were then irradiated with 6 Gy. The green and red fluorescence intensity was continuously acquired by an IncuCyte\u0026reg; S3 Live-Cell Analysis System (Sartorius, G\u0026ouml;ttingen, Germany) and with a confocal microscope (Leica TCS SP8). By using a 488 nm laser (GFP) and 552 nm laser (RFP) with 5x objective One planar in the middle of the spheroids (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eProtein interaction\u003c/h2\u003e \u003cp\u003eMaps showing the protein interactions of the autophagy players were generated and downloaded from STRING v12.0.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eUnless otherwise stated, all the experiments were performed in triplicate and repeated at least three times. The data were collected using Excel (Microsoft Office). Significance was calculated using the t test for paired samples. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate statistical significance (*).\u003c/p\u003e \u003c/div\u003e\n\u003cp\u003e\u003cem\u003eEthical approval\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Ethics Committee of University Hospital of Marburg (No. 123/19). Informed consent was obtained from all subjects involved in the study.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cem\u003eExpression of autophagy players in ATC patient tissue\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn addition to the several autophagy genes that transcribe proteins involved in the autophagosome synthesis process and catabolic activity, this study focused on the detection of the \u003cem\u003eBECN1\u003c/em\u003e, \u003cem\u003eMAP1LC3B\u003c/em\u003e, \u003cem\u003eUVRAG\u003c/em\u003e and \u003cem\u003eSQSTM1\u003c/em\u003e genes, which are responsible for autophagosome vesicle nucleation, maturation and elongation, as previously described (13, 18). Additionally, the study focused on the detection of the transcription factor EB (\u003cem\u003eTFEB\u003c/em\u003e), which is responsible for the transcription of autophagy genes, and the detection of \u003cem\u003ePRKAA1_1\u0026nbsp;\u003c/em\u003eand \u003cem\u003ePRKAA2_1\u003c/em\u003e, the two genes responsible for the transcription of the two subunits of the kinase AMPKa\u0026nbsp;(cyclic adenosine monophosphate kinase), which is a key element in linking metabolic signaling to autophagosome formation(18, 15). As shown in Figure 1A, the \u003cem\u003eBECN1\u003c/em\u003e transcript was detectable in all 19 patient samples included in the study, and its expression was more stable than that in human follicular thyroid cells. Furthermore, the expression levels of \u003cem\u003eMAP1LC3B\u003c/em\u003e, \u003cem\u003eUVRAG\u003c/em\u003e and \u003cem\u003eSQSTM1\u003c/em\u003e were significantly upregulated (median value 22.8-fold; 13.4-fold; 36.6-fold). Nonetheless, the \u003cem\u003eTFEB\u003c/em\u003e transcript was also significantly overexpressed (median value 56.9-fold). The transcripts for AMPKa, \u003cem\u003ePRKAA1_1\u003c/em\u003e and \u003cem\u003ePRKAA2_1\u003c/em\u003e were both overexpressed (8.9- and 10.2-fold median values). However, \u003cem\u003ePRKAA2_1\u003c/em\u003e was detectable in only 9 tumor tissue samples. The protein Beclin1 was detected by immunofluorescence in 10 patient samples. Figure 1B highlights the overall expression of Beclin1 in eight out of the 10 patients affected by ATC. Lower magnification micrographs of all 10 patients are included in Suppl. Figure 1.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePhoton irradiation induces the overexpression of autophagy-related gene transcripts\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eC643 cells, four primary tumor-derived cell lines (Patients 1-4) and human follicular epithelial thyroid cells (Nthy-ori-3-1) were photon irradiated with 4 or 6 Gy. Seven days after exposure, the expression of the \u003cem\u003eTFEB\u003c/em\u003e, \u003cem\u003eBECN1\u003c/em\u003e, \u003cem\u003eUVRAG\u003c/em\u003e, \u003cem\u003eSQSTM1\u003c/em\u003e, \u003cem\u003eMAP1LC3B\u003c/em\u003e, \u003cem\u003ePRKAA1_1\u003c/em\u003e and \u003cem\u003ePRKAA2_1\u003c/em\u003e genes was detected in all the cells. Exposure to 4 Gy caused significant overexpression of \u003cem\u003eTFEB\u003c/em\u003e and \u003cem\u003eSQSTM1\u003c/em\u003e in Nthy-ori-3-1, C643, Patient 2 and Patient 4 cells. Additionally, C643 cells were characterized by the significant overexpression of all the autophagy-related transcripts. However, Patient 1 exhibited stable expression of all the targets, and Patient 3 exhibited significant overexpression of only the \u003cem\u003eTFEB\u003c/em\u003e, \u003cem\u003eBECN1\u003c/em\u003e and \u003cem\u003eMAP1LC3B\u003c/em\u003e transcripts. Exposure to 6 Gy, similar to 4 Gy, caused significant overexpression of autophagy genes in all the cells. Notably, C643 cells were, once again, the most sensitive in terms of overexpressing the autophagy transcripts. Patient 1 cells once again exhibited stable expression of the autophagy transcripts after exposure to 6 Gy.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePhoton irradiation affects the protein level of autophagy players\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBased on the current findings showing the ability of irradiation to modulate autophagy at the transcriptional level, further experiments have focused on detecting the protein levels of autophagy factors in irradiated anaplastic thyroid cancer cells. As shown in Figure 3, exposure to 4 Gy caused the downregulation of Beclin, p62 and LC3B-I in all the anaplastic thyroid cancer cells. However, the protein level of LC3B-II was stable or slightly decreased (patient 3 cells). Nthy-ori-3-1 cells were the only cells that stably expressed all four proteins. Irradiation with 6 Gy also downregulated Beclin, p62, LC3B-I and LC3B-II in all cancer cells. Only Patient 2 cells were characterized by a stable protein level of LC3B-I and a significant increase in LC3B-II. The protein levels of the autophagy proteins were not altered after irradiation with 4 Gy in human follicular epithelial thyroid cells. Only 6 Gy irradiation caused a significant increase in Beclin and a significant decrease in p62 in the non-tumour cells.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eModulation of AMPKa after irradiation of ATC cells\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ecAMP kinase alpha is responsible for the phosphorylation of Unc-51 like autophagy activating kinase 1 (ULK1) and for signaling related to autophagosome formation. Recently, autophagy was shown to act independently of AMPKa in cancer(15, 13), and this kinase has been shown to inhibit autophagy (19) . Nonetheless, alterations in the expression of the downregulated gene \u003cem\u003ePRKAA1\u003c/em\u003e have recently been correlated with gastric and colorectal cancer risk and progression (20\u0026ndash;23) . Here, it was observed that the ATC cells and the Nthy-ori-3-1 cells exposed to 4 and 6 Gy were characterized by the downregulation of AMPKa. In particular, all the cells isolated 7 days after irradiation exhibited significant downregulation of the protein level of AMPKa, with the exception of Patient 4 cells, which exhibited stable expression. Furthermore, the active phosphorylated form of AMPKa (P-AMPKa) was downregulated by irradiation at both 4 and 6 Gy in all the cells. Thus, irradiation was able to inhibit the activity of AMPKa not only by suppressing the total protein concentration but also by downregulating its active form.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLive monitoring of the autophagy process in photon-irradiated ATCs\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eC643 cells stably transfected with MAP1LC3B-GFP-RFP were seeded in a round bottom low-attachment plate for 4 days prior to exposure to 6 Gy photon irradiation. Immediately after exposure to 6 Gy photon irradiation, the C643 spheroid fluorescence and morphology were continuously tracked with IncuCyte for 28 days. Double-labeled LC3B allows the detection of autophagic maturation and terminal degradation activity after fusion with the lysosome. The terminal fusion of autophagosome vesicles and lysosomes causes the degradation of LC3B and acid-sensitive GFP\u003cem\u003e.\u0026nbsp;\u003c/em\u003eInstead, acid-stable RFP retains its fluorescence. As shown in Figure 5A, C643 cells exhibited basal autophagy at the time of exposure to 6 Gy photon irradiation, as indicated by consistent green and red fluorescence. The fluorescence intensity increased every day, and both fluorescence colors merged, as highlighted by the yellow‒brownish color after day 6 of exposure, resulting from the combination of green and red fluorescence. After 7 days of exposure to 6 Gy, the spheroid morphology started to dismantle, losing its three-dimensional structure and becoming untightened, while the green/red fluorescence remained stable (Suppl. Video). Similar results were observed via confocal microscopy (Figure 5B). In particular, these micrographs provided more detailed evidence of the dismantling of the spheroid morphology and the increase in fluorescence. This result supports previous findings highlighting the efficacy of irradiation in promoting autophagy, especially 7 days after exposure.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eAnaplastic thyroid cancer is characterized by an extremely poor prognosis. Despite recent discoveries about the efficacy of personalized therapy based on mutation screening, patients affected by ATC still need therapy to significantly improve their survival rate. Combined therapy with lenvatinib, a tyrosine kinase inhibitor; pembrolizumab, a monoclonal antibody against PD-1; and the combination of dabrafenib, a BRAF inhibitor; and trametinib, a MAPK inhibitor, has shown promising effects on these patients (24). Unfortunately, prolonged treatment has led to relapse of the malignancy, thus affecting patient progression-free survival (25). For this reason, a second-line/adjuvant therapy is strongly needed to overcome the loss of efficacy of first-line therapy. The efficacy of radiotherapy in treating ATC has been highlighted previously (11, 26, 27). In particular, our previous study demonstrated that photon therapy is able to block the proliferation of ATC cells, thus preventing colony formation. The study could exclude the possibility of apoptosis as a cell death mechanism after irradiation. In fact, radiation can increase the protein level of PD-L1. Its inhibition mediated by the administration of atezolizumab exacerbates the inhibitory effect of radiotherapy. Thus, other cellular processes could be involved in the cellular decay prompted by photon irradiation. The current study focused on the ability of photon irradiation to promote autophagy as an alternative cell death mechanism. Autophagic cell death has been previously shown to be induced in anaplastic thyroid cancer cells by the administration of different compounds, thus revealing that this catabolic process is a promising therapeutic target for ATC (13). The combination of tyrosine kinase or pandeacetylase inhibitors with PD-L1 blockers, as well as berberine or even piperlongumine and capsaicin, has shown the ability to lead to autophagic cell death (28, 29, 13, 30). Here, it was shown for the first time that photon irradiation promoted autophagy in ATC cells. In particular, after seven days, 4 and 6 Gy irradiation caused the overexpression of the transcripts of the most related autophagy genes, \u003cem\u003eTFEB, BECN1, UVRAG, SQSTM1 MAP1LC3B, PRKAA1_1\u003c/em\u003e and \u003cem\u003ePRKAA2_1\u003c/em\u003e. Additionally, the protein levels of the autophagy players Beclin, LC3B-I, LC3B-II and p62 were significantly downregulated seven days after photon irradiation. The protein levels of AMPKa and its active phosphorylated form were also downregulated in irradiated cells. Thus, excluding definitively, its role in autophagy modulation. Similar effects of AMPKa have already been shown in pancreatic neuroendocrine neoplasia, where AKT exerts an inhibitory effect on AMPKa (15). However, these findings do not imply a negative modulation of autophagy that could be promoted at the transcriptional level by the cAMP responsive element or by the transcription factor TFEB (15). Moreover, after irradiation, autophagy continued to occur, and the process was further promoted, as shown by the increase in double fluorescence. This leads to a progressive dismantle of the ultrastructure of C643 cell-derived spheroids and ultimately cell decay. Anaplastic thyroid cancer tissue resected from patients showed significant overexpression of the autophagy genes \u003cem\u003eMAP1LC3B, SQSTM1, UVRAG, TFEB, PRKAA1_1\u003c/em\u003e and \u003cem\u003ePRKAA2_1\u003c/em\u003e as well as the protein Beclin1. Thus, ATC is characterized by stable active autophagy that can be promoted to induce cell death. This option has been previously shown for several solid cancers (31, 32, 15). Furthermore, autophagic cell death can promote cell death when prompted by small drugs and immune checkpoint inhibitors, not only in anaplastic thyroid cancer (13) but also in other solid malignancies (17, 33). Additionally, autophagy is likely involved in anaplastic lymphoma kinase (ALK)-associated cancers (34). Its modulation seems able to overcome the therapeutic resistance of several ALK-associated malignancies.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eThis study showed, for the first time, the ability of photon irradiation to sensitize anaplastic thyroid cancer cells to autophagy. Thus, cell death occurs. It is not known whether irradiation-mediated autophagy can modulate PD-L1 in ATCs, as previously shown (35). This ability could lead to a potential strategy for personalized therapy to more efficiently attack anaplastic thyroid cancer cells and inhibit their interaction with the tumor environment and immune cells.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eATC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Anaplastic Thyroid Cancer\u003c/p\u003e\n\u003cp\u003eGFP\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Green fluorescent protein\u003c/p\u003e\n\u003cp\u003eRFP\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Red fluorescent protein\u003c/p\u003e\n\u003cp\u003eLC3B\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Light Chain 3B\u003c/p\u003e\n\u003cp\u003eAMPKa\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;5\u0026apos;-AMP-activated protein kinase alpha\u003c/p\u003e\n\u003cp\u003eIMRT\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Intensity-modulated radiotherapy\u003c/p\u003e\n\u003cp\u003eDSBs\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Double Strand Breaks\u003c/p\u003e\n\u003cp\u003eICI\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Immune\u0026nbsp;checkpoint inhibitor\u003c/p\u003e\n\u003cp\u003emKI\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;multikinase\u0026nbsp;Inhibitor\u003c/p\u003e\n\u003cp\u003eRPMI\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Roswell Park Memorial Institute\u003c/p\u003e\n\u003cp\u003eFFPE\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Formalin Fixed Paraffin Embedded\u003c/p\u003e\n\u003cp\u003ePDTT\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Patient-Derived Tumor Tissue\u003c/p\u003e\n\u003cp\u003ePBS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Phosphate-buffered saline\u003c/p\u003e\n\u003cp\u003eFBS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Fetal bovine serum\u003c/p\u003e\n\u003cp\u003eBSA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Bovine Serum Albumin\u003c/p\u003e\n\u003cp\u003eUVRAG\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;UV Radiation Resistance-Associated Gene\u003c/p\u003e\n\u003cp\u003eSQSTM1\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Sequestosome 1\u003c/p\u003e\n\u003cp\u003eTFEB\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Transcription Factor EB\u003c/p\u003e\n\u003cp\u003ePRKAA1_1\u0026nbsp; \u0026nbsp; \u0026nbsp;5\u0026apos;-AMP-activated protein kinase catalytic subunit alpha-1\u003c/p\u003e\n\u003cp\u003ePRKAA2_1\u0026nbsp; \u0026nbsp; \u0026nbsp;5\u0026apos;-AMP-activated protein kinase catalytic subunit alpha-2\u003c/p\u003e\n\u003cp\u003eGAPDH\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Glyceraldehyde 3-Phosphate Dehydrogenase\u003c/p\u003e\n\u003cp\u003eMAP1LC3B\u0026nbsp; \u0026nbsp;\u0026nbsp;Microtubule-associated protein 1A/1B light chain 3B\u003c/p\u003e\n\u003cp\u003eBECN1\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Beclin1\u003c/p\u003e\n\u003cp\u003ePD-1\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Programmed cell death protein 1\u003c/p\u003e\n\u003cp\u003ePD-L1\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Programmed death-ligand 1\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThe study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Ethics Committee of University Hospital of Marburg (No. 123/19). Informed consent was obtained from all subjects involved in the study.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis research was funded by an MIT Research Grant from Philipps University Marburg. Open access funding was provided by the Open Access Publishing Fund of Philipps University Marburg.\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; contributions\u003c/p\u003e\n\u003cp\u003eS.W. and D.K.B. performed the surgical resection and collected the material. F.K., S.R., N.K.-G., K.R. and P.D. performed the experiments. P.D. analyzed the data. S.W. and P.D. wrote the manuscript draft. D.K.B. revised the manuscript.\u003c/p\u003e\n\u003cp\u003eAcknowledgement\u003c/p\u003e\n\u003cp\u003eWe are thankful to Stephanie Preising, Ulrike Theiss, Ekkehard Dikomey and the Institute of Radiation Biology and Molecular Radiation Oncology, Philipps University Marburg, for their technical support and use of the irradiation cabinet.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBible KC, Kebebew E, Brierley J, Brito JP, Cabanillas ME, Clark TJ, et al. 2021 American Thyroid Association Guidelines for Management of Patients with Anaplastic Thyroid Cancer. Thyroid. 2021;31(3):337\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmallridge RC, Copland JA. Anaplastic thyroid carcinoma: pathogenesis and emerging therapies. Clin Oncol (R Coll Radiol). 2010;22(6):486\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaddad RI, Lydiatt WM, Ball DW, Busaidy NL, Byrd D, Callender G, et al. Anaplastic Thyroid Carcinoma, Version 2.2015. J Natl Compr Canc Netw. 2015;13(9):1140\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eW\u0026auml;chter S, Vorl\u0026auml;nder C, Schabram J, Mintziras I, F\u0026uuml;lber I, Manoharan J, et al. Anaplastic thyroid carcinoma: changing trends of treatment strategies and associated overall survival. Eur Arch Otorhinolaryngol. 2020;277(5):1507\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePezzi TA, Mohamed ASR, Sheu T, Blanchard P, Sandulache VC, Lai SY, et al. Radiation therapy dose is associated with improved survival for unresected anaplastic thyroid carcinoma: Outcomes from the National Cancer Data Base. Cancer. 2017;123(9):1653\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFan D, Ma J, Bell AC, Groen AH, Olsen KS, Lok BH, et al. Outcomes of multimodal therapy in a large series of patients with anaplastic thyroid cancer. Cancer. 2020;126(2):444\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKwon J, Kim BH, Jung H-W, Besic N, Sugitani I, Wu H-G. The prognostic impacts of postoperative radiotherapy in the patients with resected anaplastic thyroid carcinoma: A systematic review and meta-analysis. Eur J Cancer. 2016;59:34\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaymart MR, Banerjee M, Yin H, Worden F, Griggs JJ. Marginal treatment benefit in anaplastic thyroid cancer. Cancer. 2013;119(17):3133\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin B, Ma H, Ma M, Zhang Z, Sun Z, Hsieh I-Y, et al. The incidence and survival analysis for anaplastic thyroid cancer: a SEER database analysis. Am J Transl Res. 2019;11(9):5888\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eManiakas A, Dadu R, Busaidy NL, Wang JR, Ferrarotto R, Lu C, et al. Evaluation of Overall Survival in Patients With Anaplastic Thyroid Carcinoma, 2000\u0026ndash;2019. JAMA Oncol. 2020;6(9):1397\u0026ndash;404.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eW\u0026auml;chter S, Roth S, Gercke N, Sch\u0026ouml;tz U, Dikomey E, Engenhart-Cabillic R et al. Anti-Proliferative Effect of Radiotherapy and Implication of Immunotherapy in Anaplastic Thyroid Cancer Cells. Life (Basel) 2023; 13(6).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaier P, Hartmann L, Wenz F, Herskind C. Cellular Pathways in Response to Ionizing Radiation and Their Targetability for Tumor Radiosensitization. Int J Mol Sci 2016; 17(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eW\u0026auml;chter S, Knauff F, Roth S, Keber C, Holzer K, Manoharan J, et al. Synergic Induction of Autophagic Cell Death in Anaplastic Thyroid Carcinoma. Cancer Invest. 2023;41(4):405\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eW\u0026auml;chter S, Wunderlich A, Roth S, Mintziras I, Maurer E, Hoffmann S et al. Individualised Multimodal Treatment Strategies for Anaplastic and Poorly Differentiated Thyroid Cancer. J Clin Med 2018; 7(5).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatrood S, de Prisco N, Wissniowski TT, Wiese D, Jabari S, Griesmann H, et al. Modulation of Pancreatic Neuroendocrine Neoplastic Cell Fate by Autophagy-Mediated Death. Neuroendocrinology. 2021;111(10):965\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKimura S, Noda T, Yoshimori T. Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy. 2007;3(5):452\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDi Fazio P, Waldegger P, Jabari S, Lingelbach S, Montalbano R, Ocker M, et al. Autophagy-related cell death by pan-histone deacetylase inhibition in liver cancer. Oncotarget. 2016;7(20):28998\u0026ndash;9010.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDi Fazio P, Matrood S. Targeting autophagy in liver cancer. Transl Gastroenterol Hepatol. 2018;3:39.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark J-M, Kim D-H. A paradigm shift: AMPK negatively regulates ULK1 activity. Autophagy 2023:1\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee SJ, Kang BW, Chae YS, Kim HJ, Park SY, Park JS, et al. Genetic variations in STK11, PRKAA1, and TSC1 associated with prognosis for patients with colorectal cancer. Ann Surg Oncol. 2014;21(Suppl 4):S634\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMocellin S, Verdi D, Pooley KA, Nitti D. Genetic variation and gastric cancer risk: a field synopsis and meta-analysis. Gut. 2015;64(8):1209\u0026ndash;19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen M, Jiang B, He B, Tang M, Wang P, Chen L, et al. Genetic variations in PRKAA1 predict the risk and progression of gastric Cancer. BMC Cancer. 2018;18(1):923.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu S, Tu R, Chen Z, Song J, Li P, Hu F, et al. Association of PTGER4 and PRKAA1 genetic polymorphisms with gastric cancer. BMC Med Genomics. 2023;16(1):209.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCabanillas ME, Ryder M, Jimenez C. Targeted Therapy for Advanced Thyroid Cancer: Kinase Inhibitors and Beyond. Endocr Rev. 2019;40(6):1573\u0026ndash;604.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang L, Feng Q, Wang J, Tan Z, Li Q, Ge M. Molecular basis and targeted therapy in thyroid cancer: Progress and opportunities. Biochim Biophys Acta Rev Cancer. 2023;1878(4):188928.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou W, Yue Y, Zhang X. Radiotherapy Plus Chemotherapy Leads to Prolonged Survival in Patients With Anaplastic Thyroid Cancer Compared With Radiotherapy Alone Regardless of Surgical Resection and Distant Metastasis: A Retrospective Population Study. Front Endocrinol (Lausanne). 2021;12:748023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Leo S, Trevisan M, Fugazzola L. Recent advances in the management of anaplastic thyroid cancer. Thyroid Res. 2020;13(1):17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi X-Z, Zhao S, Wang Y, Wang M-Y, Su S-W, Wu Y-Z, et al. Antitumor Activity of Berberine by Activating Autophagy and Apoptosis in CAL-62 and BHT-101 Anaplastic Thyroid Carcinoma Cell Lines. Drug Des Devel Ther. 2023;17:1889\u0026ndash;906.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin T-H, Kuo C-H, Zhang Y-S, Chen P-T, Chen S-H, Li Y-Z et al. Piperlongumine Induces Cellular Apoptosis and Autophagy via the ROS/Akt Signaling Pathway in Human Follicular Thyroid Cancer Cells. Int J Mol Sci 2023; 24(9).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu L, Xu S, Cheng X, Zhang L, Wang Y, Wu J, et al. Capsaicin inhibits the stemness of anaplastic thyroid carcinoma cells by triggering autophagy-lysosome mediated OCT4A degradation. Phytother Res. 2022;36(2):938\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatrood S, Melms LE, Bartsch DK, Di Fazio P. The Expression of Autophagy-Associated Genes Represents a Valid Footprint for Aggressive Pancreatic Neuroendocrine Neoplasms. Int J Mol Sci 2023; 24(4).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDi Fazio P, Rusche FD, Roth S, Pehl A, W\u0026auml;chter S, Mintziras I, et al. Long Non-Coding RNA H19 Expression Correlates with Autophagy Process in Adrenocortical Carcinoma. Cancer Invest. 2022;40(3):254\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWirries A, Jabari S, Jansen EP, Roth S, Figueroa-Ju\u0026aacute;rez E, Wissniowski TT, et al. Panobinostat mediated cell death: a novel therapeutic approach for osteosarcoma. Oncotarget. 2018;9(68):32997\u0026ndash;3010.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrentzel J, Sorrentino D, Giuriato S. Targeting Autophagy in ALK-Associated Cancers. Cancers (Basel) 2017; 9(12).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarc\u0026iacute;a-P\u0026eacute;rez BE, P\u0026eacute;rez-Torres C, Baltierra-Uribe SL, Castillo-Cruz J, Castrej\u0026oacute;n-Jim\u0026eacute;nez NS. Autophagy as a Target for Non-Immune Intrinsic Functions of Programmed Cell Death-Ligand 1 in Cancer. Int J Mol Sci 2023; 24(19).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"autophagy, radiotherapy, anaplastic thyroid cancer, poor prognosis, targeted therapy","lastPublishedDoi":"10.21203/rs.3.rs-5193679/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5193679/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlthough promising results have been obtained for anaplastic thyroid cancer (ATC) therapy, this therapy still needs to be improved. In addition to the currently approved therapies, radiotherapy alone or combined with adjuvant immunotherapy could be beneficial for patients affected by ATC. The patients affected by this aggressive solid malignancy could benefit from the modulation of autophagy in cancer cells. This study focused on detecting autophagy players in ATCs and bursting autophagy process via photon irradiation to induce decay in irradiated ATCs.\u003c/p\u003e \u003cp\u003eThe transcript expression of autophagy genes was detected in tumor tissue resected from 19 patients and in C643 cells, four primary ATC cell lines and primary follicular thyroid cell line (Nthy-ori-3-1) photons irradiated with 4 or 6 Gy. The protein level of Beclin1 was detected by immunofluorescence in 10/19 patients. The levels of autophagy markers were detected by RT‒qPCR and western blotting in irradiated cells. Autophagy and maturation of autophagosome vesicles were monitored in C643 cells stably transfected with the GFP-RFP-LC3B plasmid.\u003c/p\u003e \u003cp\u003eAll patients included in the study exhibited significant overexpression of autophagy transcripts. Additionally, the Beclin1 protein was expressed in resected tumor tissue. Furthermore, the analysis of autophagy-related gene transcripts revealed significant increases in the expression of these genes in C643, Patient 2 and Patient 3 cells irradiated with 4 or 6 Gy. Additionally, irradiation with 4 or 6 Gy downregulated the expression of all the proteins involved in the autophagy process. Thus, the ongoing catabolic process was confirmed. Interestingly, the levels of AMPKα and its active phosphorylated form were strongly downregulated, which excluded its involvement in autophagy activation. 6 Gy photon irradiation caused an increase in both green and red fluorescence in C643-derived spheroids. An increase in fluorescence was detectable for up to 28 days. The spheroids gradually exhibited an increase in fluorescence, which probably caused the dismantling of their ultrastructure. This finding provides evidence of the bursting of autophagy and its ability to affect spheroid integrity.\u003c/p\u003e \u003cp\u003ePhoton irradiation exacerbates autophagy in anaplastic thyroid cancer cells and could represent a valid target for focused therapy against this aggressive malignancy.\u003c/p\u003e","manuscriptTitle":"Photon Irradiation Prompts Autophagy in Anaplastic Thyroid Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-08 10:24:59","doi":"10.21203/rs.3.rs-5193679/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-04T03:36:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-03T07:17:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-03T07:16:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2024-10-02T14:55:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5e52542d-7fc2-4ed3-957f-50ac871373cc","owner":[],"postedDate":"November 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-08-31T16:05:30+00:00","versionOfRecord":{"articleIdentity":"rs-5193679","link":"https://doi.org/10.1186/s12885-026-16762-0","journal":{"identity":"bmc-cancer","isVorOnly":false,"title":"BMC Cancer"},"publishedOn":"2026-08-25 15:57:39","publishedOnDateReadable":"August 25th, 2026"},"versionCreatedAt":"2024-11-08 10:24:59","video":"","vorDoi":"10.1186/s12885-026-16762-0","vorDoiUrl":"https://doi.org/10.1186/s12885-026-16762-0","workflowStages":[]},"version":"v1","identity":"rs-5193679","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5193679","identity":"rs-5193679","version":["v1"]},"buildId":"CiT4i_kKBbxQbnFL0ufpk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}