Background
Radiotherapy (RT) represents a pivotal treatment for early and metastatic cancer. It is estimated that over half of all cancer patients can benefit from RT in combination with surgery or chemotherapy for disease management [
1,
2]. Local RT exerts its clinical effects within the irradiated (IR) field for locoregional tumor control. However, noteworthy, regression in metastatic lesions distant from IR field, albeit uncommon, has been described in patients with different types of cancer including non-small cell lung cancer (NSCLC) [
3‐
7]. This phenomenon, first described in 1953 [
8], named “abscopal effect” (AE) has been considered for many years an enigma for the scientific community. A growing body of evidence sustains the immune system activation as the dominant player in radiation-induced AE. Indeed, it has been well-established that the release of a number of moieties endowed with immunostimulatory properties, released from irradiated lesions in the tumor microenvironment and systemic circulation, are capable of conveying death messages (apoptotic and / or necrotic signals) inducing the immunogenic cell death (ICD) [
9,
10]. In particular, these molecules, also known as damage-associated molecular patterns (DAMPs), promote and convert the IR site into an immunogenic hub through the innate and adaptive immune response [
11].
Very little is known about the molecular mechanisms involved in the AE. Camphausen et al. in 2003 [
12] was the first study to link functional p53 with radiation-induced AE in mice. Accordingly, we following described AE in nude mice xenografted with wild type p53 (wtp53) colon cancer lines and receiving at least 20 Gy [
13].
Numerous cancer cell lines carrying wtp53 develop hallmarks of senescence in response to radiation or to other DNA-damaging drugs [
9,
14,
15], also known as therapy-induced senescence (TIS). TIS is highly dependent on wtp53 and p16INK4A pathways [
16,
17] and is often associated with the nuclear DNA damage response (DDR) signalling structures called DNA-SCARS [
18]. Recent literature has shed light on the importance of cytoplasmic nucleic acid sensors in DNA-damage response [
19,
20]. Indeed, when DNA is damaged, the misplaced nucleic acids into the cytoplasm engage evolutionary-conserved sensors that trigger inflammation, IFNɑ/β pathways and, as recently hypothesized, the establishment of senescence-associated pro-inflammatory secretome [
21]. Intriguingly, it was also reported that DNA:RNA hybrids structures may be largely constituted by transposable elements, in particular, long interspersed element-1 (LINE-1), the most ubiquitous transposable element in the mammalian genome, and proposed as hallmark of aging [
22].
In the present work we demonstrated that single high dose irradiation (20 Gy) induces significant tumor growth inhibition in contralateral non-irradiated (NIR) A549 but not in NIRp53-null H1299 or p53 silenced A549 (A549sh/p53) xenografts. Moreover, we provided evidences that in vitro irradiated (IR) A549 cells adopt a senescence-associated secretory phenotype (SASP) secreting CD63 positive extracellular vesicles (CD63 + EVs) loaded with DNA:RNA hybrids and LINE-1 retrotransposon inducing the senescence of distant not irradiated cells.
On the basis of these data, we hypothesized that high RT doses induce AE based on the presence of functional p53 which triggers a senescence program in cancer cells with release of senescence-inducing molecules in the tumor microenvironment and blood circulation. Our data provide proof-of-concept of a novel molecular mechanism involved in the radiation-induced AE, which could help to improve the therapeutic outcomes of patients with advanced tumor disease harboring functional p53.
Methods
Cells
A549 cells were cultured in F12K (ATCC) medium; H1299 and THP-1 (ATCC Cat# TIB-202, RRID:CVCL_0006) cells were cultured in RPMI 1640 medium; RAW 264.7 cells (ATCC Cat# TIB-71, RRID: CVCL_0493) were maintained in DMEM. All media were supplemented with 10% FBS (Euroclone, Milan, Italy) and glutamine (2 mM) (Euroclone).
Generation of A549sh/sip53 cells
Cells (3.0 × 10
4 cells/6-wells plate) were infected with either lentiviruses LV-THM-sh/scr (scrambled sh-RNA, control) or LV-THM-sh/p53 at MOI 10 TU/cell, as described [
23]. Early passages A549sh/p53 and A549sh/scr sublines were used in all experiments.
Generation of H1299p53+ cells
Cells (3.0 × 10
4 cells/6-wells plate) were transfected with p53wt expressing vector [
24] (1 μg/well) with TransIT®-LT1 Transfection Reagent (Mirus, Thermo Fisher Scientific, Milan, Italy) following the manufacturer’s guidelines. Early transfected cells (72 h after transfection) were used in all the experiments.
Mice and IR treatments
Xenograft tumours were generated in 40-days old athymic female nude mice (nu/nu CDl, Charles River Laboratories, Italy). as previously reported [
13], and IR procedures detailed in Supplemental Methods. All experiments complied with regulations and ethics guidelines of the Italian Ministry of Health and were approved by the Institutional Animal Care and Use Committee of the Regina Elena Institute (Protocol n° 366/2015-PR del 08/05/2015).
Immunohistochemical analysis
Consecutive 4-μm-thick tumor sections of formalin-fixed/paraffin-embedded xenografts were stained for Hematoxylin & Eosin (H&E), Lipofuscin detection or macrophage infiltration, as reported [
25] and detailed in
Supplementary notes.
IL6 detection
Specimens from IR, NIR or UnIR A549 xenografts were accurately selected by a pathologist for areas including at least 50% of tumour cells, then scraped-off for RNA extraction. Digital qRT-PCR (dPCR) was performed to determine IL6 expression, as described in detail in the Supplementary Methods. IL6 quantification in the sera of IR and UnIR mice was determined with xMAP multiplex technology using a Human Magnetic Luminex assay (27-plex panel, Bio-Rad Laboratories, Segrate (MI) - Italy) as described in Supplemetary notes.
In vitro IR experiments
A549, A549sh/p53 cells (as monolayer or 3D cultures), H1299, and H1299
p53+ cells, were treated with different radiation doses (10 Gy and 20 Gy) using the linear accelerator Elekta Synergy Platform system (Elekta Oncology Systems, Stockholm, Sweden) as previously reported [
26].
MTS assay
Cytotoxicity was assayed using CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Milan, Italy) according to the manufacturer’s protocol and as described in
Supplementary notes.
Senescence assay
β-galactosidase staining was performed with the Senescence β-Galactosidase Staining Kit (Cell Signaling) on 2D and 3D cell cultures according to the manufacturer’s instructions and detailed in Supplementary notes.
RNA extraction and qRT-PCR
Total RNA was extracted with TRIzol® Reagent (Invitrogen) followingmanufacturer’s instructions and qRT-PCR performed as described [
27] and detailed in Supplementary Methods. The primer sequences used are listed in
Supplemental Table 1.
Retrotransposon PCR analysis
According to established protocols [
22], purified poly(A) RNA, isolated from total RNA with NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Euroclone), was analysed with Agilent 2100 Bioanalyzer (Agilent Technologies, Milan, Italy.) to assess yield and size distribution. Then, it was reverse-transcribed (10 ng) into cDNA with Taqman kit (Applied Biosystems Italia, Monza, Italy) by replacing random primer with a strand-specific primer1. RT-qPCR was performed using SYBR Green system (Bio-Rad Laboratories), and GAPDH adopted as the normalization control.
Western blot analysis
Western Blot were performed as reported [
27] and detailed in Supplementary Methods, with following mouse monoclonal primary antibodies: anti-p21WAF1 (NeoMarkers, Fremont, CA); anti-p53 (PAb 1801) (Invitrogen, Thermo Fisher Scientific Inc., Monza, Italy). The anti-vinculin (VLN01) was used as housekeeping (Invitrogen, Thermo Fisher Scientific Inc). Quantity One Software (Bio-rad Laboratories) was used for analyses.
Confocal microscopy analysis
Cells were either fixed and permeabilized with ice-cold methanol or paraformaldehyde and incubated (overnight at 4 °C) respectively with primary anti-S9.6 antibody (1:100, Kerafast, Boston, MA, USA) or anti-CD63 antibody [MEM-259] (1:150, Abcam). Slides, after PBS washes, were incubated with the secondary goat anti-mouse Alexa Fluor 546 (Life Technologies) and confocal imaging performed with a Nikon A1 confocal laser scanning microscope.
Foci analysis procedure
Confocal images were processed for DNA:RNA hybrids, CD63+EVs and ORF1p foci quantification. The foci segmentation procedure has been developed on purpose by both MATLAB and ImageJ built-in and self-implemented functions. Details are reported in Supplementary notes.
EVs purification and characterisation
EVs used in present study were characterized according to the Minimal Information for Studies of Extracellular Vesicles guidelines, [
28] as described in the
Supplementary notes.
RNA dot blotting
Isolated EV-RNAs (200 ng/μl) were spotted onto the Hybond-N+ membrane (GE Healthcare) optimized for nucleic acid transfer. For DNA:RNA detection, membrane were UV-cross-linked andupon blocking incubated with S9.6 primary antibody (overnight at 4 °C, Kerafast), and goat anti-mouse IgG-HRP (1:5000, Santa Cruz Biotechnology Inc.) before detection by chemiluminescence with Clarity™ Western ECL Substrate (Bio-Rad Laboratories).
Macrophage polarization
Murine RAW264.7 and human THP-1 macrophage cell line were activated to M0 with M-CSF (Miltenyi Biotec, Bologna, Italy) (20 ng/ml, 48 h) or phorbol 12-myristate 13-acetate (150 nM) (Merck, Rome, Italy) respectively before to be exposed to isolated EVs The expression of M1/M2 polarization-associated markers were analysed by RT-PCR, as detailed in Supplementary notes.
Statistical analysis
All experiments were performed at least three times, and quantifiable data derived from three independent experiments and reported as mean and standard deviation. Statistical analysis for in vitro and in vivo experiments was carried out using GraphPad Prism 8 software (GraphPad Software, San Diego, CA, USA), by applying the Student t-test for 2-group comparisons. Differences were considered significant at p < 0.05. R-package was used in vivo experiments for 2-group comparisons or Kruskal-Wallis multiple comparison test as appropriate. Differences were considered statistically significant when p ≤ 0.05. The medians of foci intensity distributions were tested with a) one-sample Wilcoxon signed-rank test; b) unpaired two-sample Wilcoxon-Mann-Whitney rank test; c) unbalanced two-way ANOVA. For all the tests p-value ≤0.01 was considered for statistical significance.
Discussion
Over the years several hypotheses have been suggested to explain the molecular mechanisms behind the indirect anticancer effects of RT outside the radiation field. The difficulty in finding a single, identifiable reason for this occurrence highlights the complexity of AE and suggests that multiple pathways may contribute to triggering it. In this work, we showed that dysfunctional p53 hampers the activation of AE, suggesting only wtp53-positive cancer patients can benefit from this effect triggered by high radiation dose treatment. The presence of senescent cells and IL6 in contralateral NIR A549 tumor mass led us to investigate if the AE observed could be ascribed to the release of specific molecules from IR cells endowed with SASP.
To better clarify this phenomenon, we performed in vitro experiments aimed at emulating the in vivo irradiation condition. We showed that TP53 selectively regulates the secretion of CD63+ EVs carrying a senescence message composed of DNA:RNA hybrids and LINE-1 retrotransposons. Furthermore, the data obtained strongly support the hypothesis that CD63+ EVs together with cellular senescence, apoptosis and innate immunity may dictate the abscopal effect in NIR wtp53 A549 xenografts in nude mice. Of note, we observed that a radiation dose threshold of 10 Gy was needed for the abrogation of cytokines and the induction of DNA:RNA hybrid structures, as previously reported by Deng et al. [
31]. Furthermore, we showed that high-radiation dose in concomitantly to SASP phenotype induced in wtp53 A549 cells a large amount of cytoplasmic expression of the tetraspanin CD63, mirroring the onset of an actively secreting phenotype. The onset of SASP was accompanied by the increase of DNA:RNA hybrids in nuclear and cytoplasmic cellular compartment and in EVs. In particular, only EVs isolated from IR wtp53 A549 or from IR H1299
p53+, carried DNA:RNA hybrids that were not detectable in the EVs of p53-null H1299 or in p53-silenced A549 subjected to the same radiation treatment.
Furthermore, IR A549 or H1299p53+ EVs carrying the DNA:RNA hybrid cargo, when placed in contact with UnIR cells, induced a biomolecular make-up of recipient cells including the adoption of a senescent phenotype and reduced cell growth. These data strongly support that EVs secreted by IR cells with functional p53 are loaded with a “senescence” message.
To elucidate the factors governing the consistent macrophage recruitment observed in vivo, we exposed in vitro both murine (Fig.
7c) and human (Fig.
S3) macrophage cell lines to EVs collected from conditioned media of high-dose-IR A549 cells. Results showed that only 10 Gy-IR A549 EVs cells, carrying a DNA:RNA hybrid, induced polarization of macrophages toward the M1 phenotype.
Finally, we explored the hypothesis that DNA:RNA hybrids may be constituted by transposable elements as previously reported for senescent cells by other authors [
22]. It was previously reported that exposure to genotoxic stress such as irradiation often leads to the loss of global DNA methylation primarily from repetitive elements, in particular, LINE-1 [
32]. The activation of LINE-1 elements may induce the synthesis of high amounts of DNA:RNA hybrid structures at the nuclear level, like those described in our work, mirroring the self-retrotranscription activity of retrotransposon elements that also require a cytoplasmic step before returning to the nucleus to complete their “reproductive cycle” [
33]. Furthermore, Harris et al. [
34] reported that a large number of p53-responsive elements or p53 DNA binding sites were detected in LINE-1 elements and that at least some were functional and served to increase LINE-1 mRNA expression levels. The authors also described the triggering of a positive feedback loop where p53 activated LINE-1 transcription, further stimulating the synthesis of ORF2p and causing more DSBs and more p53 activity, which amplified the amount of DNA damage and p53-mediated DDR. Sufficient DNA damage may result in the programmed destruction of cells, reducing LINE-1 transposition (considered as DAMP molecules) in somatic cells [
34]. In our in vitro models we detected high ORF1 mRNA levels after irradiation. Furthermore, immunofluorescence experiments revealed a significant percentage of DNA:RNA ORF-1 colocalization, particularly evident in cell nuclei, which only increased in IR A549. Notably, we observed a dramatic reduction of DNA:RNA hybrids in IR A549 pre-treated with efavirenz, an anti-HIV drug that inhibits the expression of reverse transcriptase enzyme, essential for LINE-1 synthesis and its moving from one position in the genome to another via an RNA intermediate [
35]. We also observed that efavirenz pre-treatment depleted DNA:RNA hybrids in of IR A549 EVs cells abrogating their inhibitory effects on colony-forming ability of UnIR cells. All these results strongly suggest that DNA:RNA hybrids conveyed by EVs secreted by IR cells with induced SASP are largely constituted by LINE-1 retrotransposon.
Conclusion
The above observations, supported by our data, suggest that wtp53 activated by specific high radiation doses may induce the mobilization of transposon elements in both cell nuclei and cytoplasm, inducing a senescent phenotype. Functional p53 under genotoxic stress, as previously reported, plays a pivotal role in the establishment of SASP [
36,
37] and in exosome secretion, which may affect adjacent cells and immune cells [
14,
38,
39]. Our data suggest that p53 selectively regulates the secretion of CD63
+ EVs carrying a senescence message composed of DNA:RNA hybrids and LINE-1 retrotransposons (senescence-associated molecular patterns, SAMPs) that can be perceived by cells outside the field of irradiation. This, in turn, may activate auto-destruction mechanisms such as cellular senescence, apoptosis and innate immunity (Fig.
8g). However, because EVs are exquisite carriers that can target specific cells [
40] these data indeed suggest that the systemic delivery of the message may reach specifically cancer cells that belong to the same lineage as the primary tumor.
Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit
http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (
http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.