Extracellular vesicles from subjects with COPD modulate cancer initiating cells phenotype through HIF-1α shuttling

Petraroia, Ilaria; Ghidotti, Patrizia; Bertolini, Giulia; Pontis, Francesca; Roz, Luca; Balsamo, Melissa; Suatoni, Paola; Pastorino, Ugo; Ferretti, Anna Maria; Sozzi, Gabriella; Fortunato, Orazio

doi:10.1038/s41419-023-06212-1

Extracellular vesicles from subjects with COPD modulate cancer initiating cells phenotype through HIF-1α shuttling

Article
Open access
Published: 14 October 2023

Volume 14, article number 681, (2023)
Cite this article

Download PDF

You have full access to this open access article

Cell Death & Disease

Extracellular vesicles from subjects with COPD modulate cancer initiating cells phenotype through HIF-1α shuttling

Download PDF

1730 Accesses
2 Altmetric
Explore all metrics

Abstract

Chronic obstructive pulmonary disease (COPD) is a risk factor for lung cancer development. COPD induces activation of hypoxia-induced signaling, causing remodeling of surrounding microenvironmental cells also modulating the release and cargo of their extracellular vesicles (EVs). We aimed to evaluate the potential role of circulating EVs from COPD subjects in lung cancer onset. Plasma-EVs were isolated by ultracentrifugation from heavy smoker volunteers with (COPD-EVs) or without (heavy smoker-EVs, HS-EV) COPD and characterized following MISEV guidelines. Immortalized human bronchial epithelial cells (CDK4, hTERT-HBEC3-KT), genetically modified with different oncogenic alterations commonly found in lung cancer (sh-p53, KRAS^V12), were used to test plasma-EVs pro-tumorigenic activity in vitro. COPD-EVs mainly derived from immune and endothelial cells. COPD-EVs selectively increased the subset of CD133⁺CXCR4⁺ metastasis initiating cells (MICs) in HBEC-sh-p53-KRAS^V12high cells and stimulated 3D growth, migration/invasion, and acquisition of mesenchymal traits. These effects were not observed in HBEC cells bearing single oncogenic mutation (sh-p53 or KRASV12). Mechanistically, hypoxia-inducible factor 1-alpha (HIF-1α) transferred from COPD-EVs triggers CXCR4 pathway activation that in turn mediates MICs expansion and acquisition of pro-tumorigenic effects. Indeed, HIF-1α inhibition or CXCR4 silencing prevented the acquisition of malignant traits induced by COPD-EVs alone. Hypoxia recapitulates the effects observed with COPD-EVs in HBEC-sh-p53-KRAS^V12high cells. Notably, higher levels of HIF-1α were observed in EVs from COPD subjects who subsequently developed cancer compared to those who remained cancer-free. Our findings support a role of COPD-EVs to promote the expansion of MICs in premalignant epithelial cells through HIF-1α-CXCR4 axis activation thereby potentially sustaining lung cancer progression.

Circulating extracellular vesicles from individuals at high-risk of lung cancer induce pro-tumorigenic conversion of stromal cells through transfer of miR-126 and miR-320

Article Open access 21 July 2021

c-Myc shuttled by tumour-derived extracellular vesicles promotes lung bronchial cell proliferation through miR-19b and miR-92a

Article Open access 07 October 2019

Similar programmed death ligand 1 (PD-L1) expression profile in patients with mild COPD and lung cancer

Article Open access 27 December 2022

Introduction

Cigarette smoking can be considered the main risk factor for the development of chronic obstructive pulmonary disease (COPD) and for the onset of lung cancer [1]. However, only 20% of smokers develop COPD [2], whereas the incidence of lung cancer is higher in subjects with COPD [3]. Since COPD and lung cancer display the activation of some common pathways related to inflammation or growth factors [4], it is likely that the presence of COPD could be a potent driver for lung cancer progression. In NSCLC, we and other shown that CD133 represents a robust marker to identify cells endowed with stem like properties and cancer initiating potential (cancer-initiating cells-CICs) [5,6,7]. It is now well-recognized that CICs represent an heterogeneous population comprised of cells deputed to specific function, endowed with different properties and characterized by specific markers [8]. In lung cancer for instance different markers have been described to possibly identify CIC subset, suggesting a great phenotypic heterogeneity of such population [9,10,Full size image

Based on these preliminary results, we focused our attention on HBEC-KRAS^V12high in which a more striking effect in the acquisition of a malignant phenotype was observed after COPD-EVs treatment. We further observed that the treatment with COPD-EVs significantly increased the percentage of wound closure compared to HS-EVs (% wound closure: COPD-EVs= 75.8%, SEM = 5.1%; HS-EVs= 58.2%, SEM = 4.8%; NT = 60.6%, SEM = 3.1%. p = 0.0451 vs. HS-EVs and p = 0.042 vs. NT) (Fig. 3G and Supplementary Fig. S3). Instead, proliferation assay showed that COPD-EVs did not affect the growth rate of HBEC-KRAS^V12high, cultured as monolayer (Fig. 3H); however, COPD-EVs but not HS-EVs fostered the 3D-colony forming ability of HBEC-KRAS^V12high, providing and indirect indication on the pro-tumorigenic features of COPD-EVs treated cells (Fig. 3I). Overall, these results suggest that COPD-EVs promote the increase of CD133⁺ cancer-initiating cells, in particular through the expansion of CD133⁺CXCR4⁺ MICs subset, along with the acquisition of pro-tumorigenic and invasive properties in HBEC-KRAS^V12high cells.

HIF-1α is transferred from COPD-EVs to HBEC-KRAS^V12high

To investigate the factors causing the increase of MICs subset and the acquisition of pro-tumorigenic properties induced by COPD-EVs we focused on SDF-1, the cytokine that activates CXCR4 downstream signaling pathways, also known to regulate CICs phenotype [30]. Treatment of HBEC-KRAS^V12high cells with neutralizing antibody against SDF-1 did not abrogate the modulation of CICs cells induced by COPD-EVs, indicating that SDF-1-CXCR4 activation axis was not central in COPD-EVs induced expansion of CICs (Fig. 4A). Since it has been demonstrated that COPD causes a hypoxic lung microenvironment, we decided to analyze inside EVs the amount of the transcription factor HIF-1α, the master regulator of the cellular response to hypoxia. HIF-1α level, evaluated via ELISA, was significantly lower in HS-EVs (2.1 pg/ml, SEM = 1.5 pg/ml) than in COPD-EVs (7.5 pg/ml, SEM = 2.0 pg/ml) (*p = 0.03) (Fig. 4B). The transfer of HIF-1α into recipient cells via EVs was evaluated by treating HBEC-KRAS^V12high with PKH-labeled-EVs. Intracellular level of HIF-1α in PKH-fluorescent positive cells treated with COPD-EVs was 2.2-fold increased compared to the HS-EV treatment (p < 0.01) (Fig. 4C). To further elucidate whether the increase of HIF-1α in recipient cells was actually due to EVs protein shuttling or to the induction of endogenous transcript, levels of HIF-1α mRNA were evaluated after EVs treatment of HBEC-KRAS^V12high. No differences in HIF-1α transcription were observed between the two groups (Fig. 4D), indicating that HIF-1α was not induced into recipient cells but shuttled by EVs. Moreover, the transfer of HIF-1α from COPD-EVs induced the transcription of known downstream targets such as VEGF and CAIX in recipient cells (Fig. 4D). To assess the role of HIF-1α in the induction of MICs phenotype caused by COPD-EVs, we exploited a selective HIF-1α inhibitor, PX-478, before EVs treatment. HIF-1α inhibition completely abrogates the expansion of HBEC-KRAS^V12high CD133⁺ CICs induced by COPD-EVs, especially acting on CD133⁺CXCR4⁺ MICs (Fig. 4E). Coherently with effects on tumorigenic CICs subset, the blockade of HIF-1α transfer after COPD-EVs treatment reduced the number of 3D-colonies (Fig. 4F). Furthermore, the inhibition of HIF-1α reversed the pro-migratory effect, towards both SDF-1 and EGF chemoattractants, induced by COPD-EVs (Fig. 4G).

**Fig. 4: HIF-1α is transferred by COPD-EVs in recipient cells.**

HIF-1α shuttle induces CXCR4 modulation and CD133⁺ stem cells

Since HIF-1α controls directly CXCR4 expression that in turn modulates CICs phenotype, we investigated its possible involvement as mediator of COPD-EVs induced-CICs effect. We confirmed that CXCR4 transcription was significantly increased in HBEC-KRAS^V12high cells after COPD-EVs treatment and that the inhibition of HIF-1α completely abrogated this effect (Fig. 5A). Notably, no CXCR4 modulation was observed after HS-EVs administration. Furthermore, CXCR4 silencing in HBEC-KRAS^V12high recipient cells completely prevented the increase of CD133⁺ and CD133⁺CXCR4⁺ subsets caused by COPD-EVs (Fig. 5B). Of note, the blockade of CXCR4 in HBEC-KRAS^V12high reverted the capacity of COPD-EVs to promote migration (Fig. 5C) and invasion (Fig. 5D) of these cells. Furthermore, the number of 3D colonies induced by COPD-EVs was reduced after CXCR4 silencing in recipient cells (Fig. 5E).

All together these data support the crucial role of CXCR4 as a mediator of hypoxia-induced modulation of MIC phenotype.

Hypoxia plays a key role in MIC induction and in cancer development

To further demonstrate the central role of hypoxia in the regulation of MIC phenotype, HBEC-1, HBEC-3, HBEC-5, HBEC-6, and HBEC-KRAS^V12high were incubated for 72 h at 2% oxygen and the modulation of CD133⁺CXCR4⁺ was evaluated. Hypoxia induced a 4-fold increase of MIC cells only in HBEC-KRAS ^V12high cells (*p = <0.05) unlike other cell lines tested (Fig. 6A). Once again, the HIF-1α blocking (Fig. 6B) or the CXCR4 silencing (Fig. 6C) prevented the expansion of MICs in HBEC-KRAS^V12high in hypoxic conditions. Finally, we demonstrated that hypoxia promoted migratory and invasive capacities of HBEC-KRAS^V12high that are abrogated by the inhibition of HIF-1α or CXCR4 in recipient cells towards an EGF gradient (Fig. 6D, E and Supplementary Fig. S4). These results corroborate the observation that the cellular response to hypoxia is central in promoting MIC expansion but only in transformed epithelial cells accumulating multiple oncogenic hits, similar to what we previously observed after COPD-EVs treatment.

**Fig. 6: Hypoxia induces MIC expansion.**

Our MACSPlex analysis in Fig. 1E indicated a significant enrichment of endothelial markers in circulating EVs obtained from COPD subjects, suggesting their endothelial cell origin. To assess whether hypoxic environment occurring in COPD subjects promotes EVs release by endothelial cells, we cultured endothelial cells in hypoxic and standard oxygen culture conditions and we next recovered and analyzed EVs via MACSPlex. Hypoxia induced an increase in endothelial markers on the surface of EVs compared to normal condition (Fig. 6F), confirming that hypoxia boosted the release of EVs from endothelial cells.

We verified that EVs released by endothelial cells, maintained both in normoxic (END-EVs Normoxia) and in hypoxic conditions (END-EVs Hypoxia), have a similar ability to increase the total number of CD133⁺ cells in HBEC-KRAS^V12high recipient cells, but EVs released by endothelial cells under hypoxia can specifically expand the pool of CD133⁺CXCR4⁺ cells, suggesting their unique ability to activate CXCR4 pathway (Fig. 6G).

Finally, to confirm the involvement of hypoxia in cancer development, we analyzed the level of HIF-1α inside EVs from COPD volunteers, who subsequently developed or not lung cancer. We found significant higher levels of HIF-1α (125.8 pg/ml, SEM = 48.66 pg/ml) in EVs from COPD subjects who subsequently developed cancer (i.e. before CT scan detection) than COPD subjects who remained cancer free for at least three years (12.34 pg/ml, SEM = 2.835 pg/ml) (*p = 0.04) (Fig. 6H).

Taken together, these results highlight the strong involvement of COPD-derived hypoxia in inducing the release of EVs by endothelial cells and the potential role of HIF-1α within EVs in fostering lung cancer development that may be exploited as biomarker of lung cancer risk.

Discussion

Smoking leads to an increase of inflammation in the lungs that modifies the phenotype of epithelial and stromal cells [28]. COPD is associated with persistent respiratory symptoms and elevated inflammation levels [31]. Several evidences link the persistence of COPD with a higher incidence of lung cancer [32]. However, only 1 % of COPD subjects develop lung cancer indicating the existence of a complex mechanism behind the increased risk of lung carcinogenesis [33].

Recently, EVs emerged as important regulators of COPD and lung cancer [34]. It has been reported that COPD and inflammation modifies the phenotype of circulating EVs either considering surface markers or cargo [23]. Indeed, this pathological condition stimulates the release of EVs from endothelial cells and monocytes in the blood [35], whereas in the broncho-alveolar lavage vesicles expressing endothelial and macrophages markers were detected in COPD smokers compared to non smoker subjects [36]. In both of these studies the amount of vesicles correlates with the severity of COPD. Here, we confirmed a higher presence of endothelial-derived EVs in COPD subjects but we also found a general increase of surface markers derived from immune cells suggesting a possible involvement of adaptive immune response in the pathogenesis of the disease.

Hypoxia-inducible factors (HIFs), strongly deregulated in COPD disease, were also deregulated in several solid tumors and modulated different signaling pathways such as proliferation, EMT, metastatization and also resistance to therapy [37]. It has been demonstrated in experimental mouse models that COPD-like airway inflammation can lead to lung cancer promotion in a context of KRAS mutant epithelial cells [38, 39]. Interestingly, we proved that both in vitro hypoxic conditions and HIF-1α shuttled from COPD-EVs to recipient cells induce aggressive cancer phenotype and malignant progression through the increase of MICs population, but only in the context of an already activated genetic background of epithelial cells carrying different oncogenic hits, including amplification of KRAS^V12.

HIF-1α and hypoxia are involved in EV biogenesis both in normal and pathological conditions and are also implicated in the regulation of EV cargo such as protein and miRNAs [40]. It has been shown that nasopharyngeal carcinoma-derived vesicles induced EMT phenotype in recipient cell through the direct transfer of HIF-1α and this modulation increase the migratory and pro-metastatic ability of these cells [41]. Moreover, HIF-1α directly regulates the transcription of CXCR4, a chemokine receptor involved in cancer cell migration, invasion and in the maintenance of CICs in different solid tumors [42,

Materials and methods

Cell lines

Immortalized human bronchial epithelial cells (HBEC-1: hTERT+Cdk4; HBEC-3: hTERT+Cdk4 + KRAS^V12; HBEC-5: hTERT+Cdk4+sh-p53; HBEC-6: hTERT+Cdk4+sh-p53 + KRAS^V12; HBEC-KRAS^V12high: hTERT+Cdk4+sh-p53+KRAS^V12high) were provided by Prof. J.D. Minna (UT Southwestern, TX) [46]. Cells were cultured in Keratinocyte–Serum-Free Medium supplemented with 5 ng/ml human recombinant EGF and 50 μg/ml bovine pituitary extract (K-SFM; Thermo Fisher Scientific, Waltham Massachusetts, USA).

More details on cells treatments are provided in Supplementary Methods.

Clinical specimens

Plasma samples were collected from heavy smoker volunteers (age 50–75 years) enrolled in a low dose CT screening trial performed at our institution (BioMild Trial, ClinicalTrials.gov: NCT02247453) [47]. Clinical characteristics of heavy smokers volunteers cancer free and heavy smokers volunteers who developed lung cancer are summarized in Table 1. Plasma collection was approved by Internal Review and Ethics Boards of Istituto Nazionale Tumori of Milan (INT 11-21).

Table 1 Clinical characteristics of heavy-smokers volunteers.

Full size table

EVs isolation and characterization

EVs were isolated from the plasma of heavy smokers and COPD volunteers by ultracentrifugation at 120,000 × g at 4 °C for 90 min using a TLA-100.3 fixed-angle rotor in a TL-100 ultracentrifuge (Beckman Coulter, Brea, CA, USA). EV-enriched pellet was washed in filtered phosphate-buffered saline (PBS; Thermo Fisher Scientific) centrifuged at 120,000 × g for 60 min at 4 °C to eliminate impurities and later resuspended in PBS. The protein content of purified EVs was verified by Bradford assay.

For EVs isolation from conditioned media (CM), HUVEC were maintained in serum-free medium for 72 h in normal oxygen condition or in 2 % oxygen condition at 37 °C. Then, the CM was collected and centrifuged at 3200 × g for 25 min in order to remove cell debris. EVs isolation was performed following the same protocol used for plasma.

EVs concentration and size distribution were evaluated by using a NanoSight NS300 instrument (Malvern Panalytical) whereas surface markers profile of plasma EVs was characterized by MACSPlex Kit (Milteny Biotec), as detailed in Supplementary Methods.

Flow cytometry

EVs analysis

EVs (15 µg) were incubated with 10 µl of Aldehyde/Sulfate latex beads, 4% w/v 4 µm (Invitrogen, Thermo Fisher Scientific) for 15 min at room temperature (RT) and later incubated overnight at 4 °C in PBS. After incubation, 100 mM glycine was added for 30 min at RT and then samples were stained with antibodies, as detailed in Supplementary Methods.

HBEC analysis

Quantification of CICs in different cell lines was performed 72 h post-treatment with EVs. Cells were resuspended in staining buffer (PBS 1X + 0.5 % BSA) and incubated with PE-conjugate anti-human CD133 antibody (Miltenyi Biotec) and APC-conjugate anti-human CXCR4 (BD Pharmingen) antibody for 20 min at 4 °C. After washing, cells were incubated with 7-AAD live/dead solution (Thermo Fisher Scientific) and the acquisition was performed using a FACSCanto II flow cytometer (BD Pharmingen).

Wound-healing assay

Wound-healing assay was performed to evaluate the migratory ability of HBEC-KRAS^V12high. 5×10⁴ HBEC-KRAS^V12high, pre-treated with 15 µg of HS- or COPD-EVs, were seeded on both sides of Culture Insert 2-well in µ-Dish 35 mm (Ibidi). After 24 h, the culture insert was removed and 1 ml of Keratynocyte-SFM was added in each well. The cell-free gap was monitored and photographed immediately and after 3 h using a JuLI Stage Real-Time Cell History Recorder (NanoEntek). The percentage of wound closure was evaluated in 5 random fields using the ImageJ Software.

3D Proliferation assay

The 3D proliferation assay was performed to evaluate the ability of HBEC-KRAS^V12high to grow as colonies. The assay was performed using Vitrogel 3D (TheWell Bioscience, USA). A 1:1 VitroGel mix was added to 24-well plates and incubated for 20 min at 37 °C. Then, 5 × 10⁴ of pretreated HBEC-KRAS^V12high were seeded on the VitroGel’s layer. After 14 days, the number of colonies was counted in 5 random fields per condition.

Statistical analysis

The statistical analysis was performed using the Graphpad Prism 5 software. Statistically significant differences were determined with Student’s t‐test when comparing two groups or the ANOVA test for multiple comparisons.

Data availability

All datasets generated and analyzed during this study are included in this published article and its Supplementary Information files. Additional data are available from the corresponding author on reasonable request.

References

Blanco I, Diego I, Bueno P, Casas-Maldonado F, Miravitlles M. Geographic distribution of COPD prevalence in the world displayed by Geographic Information System maps. Eur Respir J. 2019;54:1900610.
Terzikhan N, Verhamme KM, Hofman A, Stricker BH, Brusselle GG, Lahousse L. Prevalence and incidence of COPD in smokers and non-smokers: the Rotterdam Study. Eur J Epidemiol. 2016;31:785–92.
Article CAS PubMed PubMed Central Google Scholar
Durham AL, Adcock IM. The relationship between COPD and lung cancer. Lung Cancer. 2015;90:121–7.
Article CAS PubMed Google Scholar
Moermans C, Heinen V, Nguyen M, Henket M, Sele J, Manise M, et al. Local and systemic cellular inflammation and cytokine release in chronic obstructive pulmonary disease. Cytokine. 2011;56:298–304.
Article CAS PubMed Google Scholar
Eramo A, Lotti F, Sette G, Pilozzi E, Biffoni M, Di Virgilio A, et al. Identification and expansion of the tumorigenic lung cancer stem cell population. Cell Death Differ. 2008;15:504–14.
Article CAS PubMed Google Scholar
Bertolini G, Roz L, Perego P, Tortoreto M, Fontanella E, Gatti L, et al. Highly tumorigenic lung cancer CD133+ cells display stem-like features and are spared by cisplatin treatment. Proc Natl Acad Sci USA. 2009;106:16281–6.
Article CAS PubMed PubMed Central Google Scholar
Tirino V, Camerlingo R, Franco R, Malanga D, La RA, Viglietto G, et al. The role of CD133 in the identification and characterisation of tumour-initiating cells in non-small-cell lung cancer. Eur J Cardiothorac Surg. 2009;36:446–53.
Article PubMed Google Scholar
Prasetyanti PR, Medema JP. Intra-tumor heterogeneity from a cancer stem cell perspective. Mol Cancer. 2017;16:41.
Article PubMed PubMed Central Google Scholar
Sullivan JP, Spinola M, Dodge M, Raso MG, Behrens C, Gao B, et al. Aldehyde dehydrogenase activity selects for lung adenocarcinoma stem cells dependent on notch signaling. Cancer Res. 2010;70:9937–48.
Article CAS PubMed PubMed Central Google Scholar
Leung EL, Fiscus RR, Tung JW, Tin VP, Cheng LC, Sihoe AD, et al. Non-small cell lung cancer cells expressing CD44 are enriched for stem cell-like properties. PLoS ONE. 2010;5:e14062.
Article PubMed PubMed Central Google Scholar
Liu J, **ao Z, Wong SK, Tin VP, Ho KY, Wang J, et al. Lung cancer tumorigenicity and drug resistance are maintained through ALDH(hi)CD44(hi) tumor initiating cells. Oncotarget. 2013;4:1698–711.
Article PubMed PubMed Central Google Scholar
Zhang WC, Shyh-Chang N, Yang H, Rai A, Umashankar S, Ma S, et al. Glycine decarboxylase activity drives non-small cell lung cancer tumor-initiating cells and tumorigenesis. Cell. 2012;148:259–72.
Article CAS PubMed Google Scholar
Bertolini G, D’Amico L, Moro M, Landoni E, Perego P, Miceli R, et al. Microenvironment-modulated metastatic CD133+/CXCR4+/EpCAM-lung cancer-initiating cells sustain tumor dissemination and correlate with poor prognosis. Cancer Res. 2015;75:3636–49.
Article CAS PubMed Google Scholar
Genschmer KR, Russell DW, Lal C, Szul T, Bratcher PE, Noerager BD, et al. Activated PMN exosomes: pathogenic entities causing matrix destruction and disease in the lung. Cell. 2019;176:113–26.
Article CAS PubMed PubMed Central Google Scholar
Vlahos R, Bozinovski S. Role of alveolar macrophages in chronic obstructive pulmonary disease. Front Immunol. 2014;5:435.
Article PubMed PubMed Central Google Scholar
Pullamsetti SS, Mamazhakypov A, Weissmann N, Seeger W, Savai R. Hypoxia-inducible factor signaling in pulmonary hypertension. J Clin Invest. 2020;130:5638–51.
Article CAS PubMed PubMed Central Google Scholar
Wu H, Ma H, Wang L, Zhang H, Lu L, **ao T, et al. Regulation of lung epithelial cell senescence in smoking-induced COPD/emphysema by microR-125a-5p via Sp1 mediation of SIRT1/HIF-1a. Int J Biol Sci. 2022;18:661–74.
Article CAS PubMed PubMed Central Google Scholar
Zhang Q, Han Z, Zhu Y, Chen J, Li W. Role of hypoxia inducible factor-1 in cancer stem cells (Review). Mol Med Rep. 2021;23:17.
Korbecki J, Kojder K, Kapczuk P, Kupnicka P, Gawrońska-Szklarz B, Gutowska I. The effect of hypoxia on the expression of CXC chemokines and CXC chemokine receptors—a review of literature. Int J Mol Sci. 2021;22:843.
Article CAS PubMed PubMed Central Google Scholar
Mohan A, Agarwal S, Clauss M, Britt NS, Dhillon NK. Extracellular vesicles: novel communicators in lung diseases. Respir Res. 2020;21:175.
Article PubMed PubMed Central Google Scholar
Herrmann A, Rice M, LÃ©vy R, Pizer BL, Losty PD, Moss D, et al. Cellular memory of hypoxia elicits neuroblastoma metastasis and enables invasion by non-aggressive neighbouring cells. Oncogenesis. 2015;4:e138.
Article CAS PubMed PubMed Central Google Scholar
Zonneveld MI, Keulers TGH, Rouschop KMA. Extracellular vesicles as transmitters of hypoxia tolerance in solid cancers. Cancers (Basel). 2019;11:154.
O’Farrell HE, Bowman RV, Fong KM, Yang IA. Plasma extracellular vesicle miRNAs can identify lung cancer, current smoking status, and stable COPD. Int J Mol Sci. 2021;22:5803.
Cañas JA, Sastre B, Rodrigo-Muñoz JM, Del Pozo V. Exosomes: a new approach to asthma pathology. Clin Chim Acta. 2019;495:139–47.
Article PubMed Google Scholar
Kadota T, Fujita Y, Yoshioka Y, Araya J, Kuwano K, Ochiya T. Extracellular vesicles in chronic obstructive pulmonary disease. Int J Mol Sci. 2016;17:1801.
Li M, Yu D, Williams KJ, Liu ML. Tobacco smoke induces the generation of procoagulant microvesicles from human monocytes/macrophages. Arterioscler Thromb Vasc Biol. 2010;30:1818–24.
Article CAS PubMed PubMed Central Google Scholar
Kubo H. Extracellular vesicles in lung disease. Chest. 2018;153:210–6.
Article PubMed Google Scholar
Feller D, Kun J, Ruzsics I, Rapp J, Sarosi V, Kvell K, et al. Cigarette smoke-induced pulmonary inflammation becomes systemic by circulating extracellular vesicles containing Wnt5a and inflammatory cytokines. Front Immunol. 2018;9:1724.
Article PubMed PubMed Central Google Scholar
ThÃ©ry C, Witwer KW, Aikawa E, Alcaraz MJ, Anderson JD, Andriantsitohaina R, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. 2018;7:1535750.
Article Google Scholar
Teicher BA, Fricker SP. CXCL12 (SDF-1)/CXCR4 pathway in cancer. Clin Cancer Res. 2010;16:2927–31.
Article CAS PubMed Google Scholar
Oudijk EJ, Lammers JW, Koenderman L. Systemic inflammation in chronic obstructive pulmonary disease. Eur Respir J Suppl. 2003;46:5s–13s.
Article CAS PubMed Google Scholar
Sekine Y, Katsura H, Koh E, Hiroshima K, Fujisawa T. Early detection of COPD is important for lung cancer surveillance. Eur Respir J. 2012;39:1230–40.
Article PubMed Google Scholar
Parris BA, O’Farrell HE, Fong KM, Yang IA. Chronic obstructive pulmonary disease (COPD) and lung cancer: common pathways for pathogenesis. J Thorac Dis. 2019;11:S2155–S2172.
Article PubMed PubMed Central Google Scholar
Szalontai K, Gémes N, Furák J, Varga T, Neuperger P, Balog JÁ, et al. Chronic obstructive pulmonary disease: epidemiology, biomarkers, and paving the way to lung cancer. J Clin Med. 2021;10:2889.
Article CAS PubMed PubMed Central Google Scholar
Nieri D, Daniele M, Lombardi S, Bazzan E, Santerini S, De Cusatis G, et al. Circulating extracellular vesicles are associated with disease severity and interleukin-6 levels in COPD: a pilot study. J Clin Med. 2021;10:5014.
Bazzan E, Radu CM, TinÃ¨ M, Neri T, Biondini D, Semenzato U, et al. Microvesicles in bronchoalveolar lavage as a potential biomarker of COPD. Am J Physiol Lung Cell Mol Physiol. 2021;320:L241–L245.
Article CAS PubMed Google Scholar
Hapke RY, Haake SM. Hypoxia-induced epithelial to mesenchymal transition in cancer. Cancer Lett. 2020;487:10–20.
Article CAS PubMed PubMed Central Google Scholar
Moghaddam SJ, Li H, Cho SN, Dishop MK, Wistuba II, Ji L, et al. Promotion of lung carcinogenesis by chronic obstructive pulmonary disease-like airway inflammation in a K-ras-induced mouse model. Am J Respir Cell Mol Biol. 2009;40:443–53.
Article CAS PubMed Google Scholar
Gong L, Cumpian AM, Caetano MS, Ochoa CE, De la Garza MM, Lapid DJ, et al. Promoting effect of neutrophils on lung tumorigenesis is mediated by CXCR2 and neutrophil elastase. Mol Cancer. 2013;12:154.
Article PubMed PubMed Central Google Scholar
Kucharzewska P, Christianson HC, Welch JE, Svensson KJ, Fredlund E, RingnÃ©r M, et al. Exosomes reflect the hypoxic status of glioma cells and mediate hypoxia-dependent activation of vascular cells during tumor development. Proc Natl Acad Sci USA. 2013;110:7312–7.
Article CAS PubMed PubMed Central Google Scholar
Aga M, Bentz GL, Raffa S, Torrisi MR, Kondo S, Wakisaka N, et al. Exosomal HIF1Î± supports invasive potential of nasopharyngeal carcinoma-associated LMP1-positive exosomes. Oncogene. 2014;33:4613–22.
Article CAS PubMed PubMed Central Google Scholar
Romain B, Hachet-Haas M, Rohr S, Brigand C, Galzi JL, Gaub MP, et al. Hypoxia differentially regulated CXCR4 and CXCR7 signaling in colon cancer. Mol Cancer. 2014;13:58.
Article PubMed PubMed Central Google Scholar
Bao Y, Wang Z, Liu B, Lu X, **ong Y, Shi J, et al. A feed-forward loop between nuclear translocation of CXCR4 and HIF-1Î± promotes renal cell carcinoma metastasis. Oncogene. 2019;38:881–95.
Article CAS PubMed Google Scholar
Borzi C, Calzolari L, Ferretti AM, Caleca L, Pastorino U, Sozzi G, et al. c-Myc shuttled by tumour-derived extracellular vesicles promotes lung bronchial cell proliferation through miR-19b and miR-92a. Cell Death Dis. 2019;10:759.
Article PubMed PubMed Central Google Scholar
Pontis F, Roz L, Mensah M, Segale M, Moro M, Bertolini G, et al. Circulating extracellular vesicles from individuals at high-risk of lung cancer induce pro-tumorigenic conversion of stromal cells through transfer of miR-126 and miR-320. J Exp Clin Cancer Res. 2021;40:237.
Article CAS PubMed PubMed Central Google Scholar
Sato M, Larsen JE, Lee W, Sun H, Shames DS, Dalvi MP, et al. Human lung epithelial cells progressed to malignancy through specific oncogenic manipulations. Mol Cancer Res. 2013;11:638–50.
Article CAS PubMed PubMed Central Google Scholar
Pastorino U, Boeri M, Sestini S, Sabia F, Milanese G, Silva M, et al. Baseline computed tomography screening and blood microRNA predict lung cancer risk and define adequate intervals in the BioMILD trial. Ann Oncol. 2022;33:395–405.

Download references

Funding

The study was supported by grants from the Italian Association for Cancer Research (Investigator Grant 23244 to G.S.; 21431 to L.R.), Italian Ministry of Health (RF-2018-12367824 to G.S.; GR-2019-12369047 to O.F; RF-2016-02362946 to L.R.), Fondazione Regionale per la Ricerca Biomedica (Regione Lombardia) (1731093 to G.B.), Italian Ministry of Health “Ricerca Corrente” funds.

Author information

These authors contributed equally: Ilaria Petraroia, Patrizia Ghidotti.
These authors jointly supervised this work: Gabriella Sozzi, Orazio Fortunato.

Authors and Affiliations

Epigenomics and biomarkers of solid tumors, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy
Ilaria Petraroia, Patrizia Ghidotti, Giulia Bertolini, Francesca Pontis, Luca Roz, Melissa Balsamo, Gabriella Sozzi & Orazio Fortunato
Thoracic Surgery Unit, Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy
Paola Suatoni & Ugo Pastorino
Istituto di Scienze e Tecnologie Chimiche-CNR, Milano, Italy
Anna Maria Ferretti

Authors

Ilaria Petraroia
View author publications
You can also search for this author in PubMed Google Scholar
Patrizia Ghidotti
View author publications
You can also search for this author in PubMed Google Scholar
Giulia Bertolini
View author publications
You can also search for this author in PubMed Google Scholar
Francesca Pontis
View author publications
You can also search for this author in PubMed Google Scholar
Luca Roz
View author publications
You can also search for this author in PubMed Google Scholar
Melissa Balsamo
View author publications
You can also search for this author in PubMed Google Scholar
Paola Suatoni
View author publications
You can also search for this author in PubMed Google Scholar
Ugo Pastorino
View author publications
You can also search for this author in PubMed Google Scholar
Anna Maria Ferretti
View author publications
You can also search for this author in PubMed Google Scholar
Gabriella Sozzi
View author publications
You can also search for this author in PubMed Google Scholar
Orazio Fortunato
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

IP, PG, FP, MB performed all the experiments under the supervision of OF and GB; AMF performed electronic microscopy analysis; OF, GB, LR, GS conceived the project and designed the experiments; OF, GB analyzed and interpreted the data; UP run the lung cancer screening trial, enrolled volunteers and collected clinical information; PS collected subjects samples; OF, GB, LR, GS wrote the manuscript. All authors reviewed and approved the manuscript.

Corresponding author

Correspondence to Giulia Bertolini.

Ethics declarations

Competing interests

The authors declare no competing interests.

Ethics

Human samples were obtained under protocols approved by the Independent Ethics Committee of the Istituto Nazionale dei Tumori (INT 11-21).

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Edited by Dr Giovanni Blandino

Supplementary information

Supplementary materials

Reproducibility Checklist

Rights and permissions

Open Access This 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Petraroia, I., Ghidotti, P., Bertolini, G. et al. Extracellular vesicles from subjects with COPD modulate cancer initiating cells phenotype through HIF-1α shuttling. Cell Death Dis 14, 681 (2023). https://doi.org/10.1038/s41419-023-06212-1

Download citation

Received: 27 April 2023
Revised: 25 September 2023
Accepted: 03 October 2023
Published: 14 October 2023
DOI: https://doi.org/10.1038/s41419-023-06212-1
Springer Nature Limited

Extracellular vesicles from subjects with COPD modulate cancer initiating cells phenotype through HIF-1α shuttling

Abstract

Similar content being viewed by others

Circulating extracellular vesicles from individuals at high-risk of lung cancer induce pro-tumorigenic conversion of stromal cells through transfer of miR-126 and miR-320

c-Myc shuttled by tumour-derived extracellular vesicles promotes lung bronchial cell proliferation through miR-19b and miR-92a

Similar programmed death ligand 1 (PD-L1) expression profile in patients with mild COPD and lung cancer

Introduction

HIF-1α is transferred from COPD-EVs to HBEC-KRAS^V12high

HIF-1α shuttle induces CXCR4 modulation and CD133⁺ stem cells

Hypoxia plays a key role in MIC induction and in cancer development

Discussion

Materials and methods

Cell lines

Clinical specimens

EVs isolation and characterization

Flow cytometry

EVs analysis

HBEC analysis

Wound-healing assay

3D Proliferation assay

Statistical analysis

Data availability

References

Funding

Author information

Authors and Affiliations

Contributions

Corresponding author

Ethics declarations

Competing interests

Ethics

Additional information

Supplementary information

Supplementary materials

Reproducibility Checklist

Rights and permissions

About this article

Cite this article

Navigation

Extracellular vesicles from subjects with COPD modulate cancer initiating cells phenotype through HIF-1α shuttling

Abstract

Similar content being viewed by others

Circulating extracellular vesicles from individuals at high-risk of lung cancer induce pro-tumorigenic conversion of stromal cells through transfer of miR-126 and miR-320

c-Myc shuttled by tumour-derived extracellular vesicles promotes lung bronchial cell proliferation through miR-19b and miR-92a

Similar programmed death ligand 1 (PD-L1) expression profile in patients with mild COPD and lung cancer

Introduction

HIF-1α is transferred from COPD-EVs to HBEC-KRASV12high

HIF-1α shuttle induces CXCR4 modulation and CD133+ stem cells

Hypoxia plays a key role in MIC induction and in cancer development

Discussion

Materials and methods

Cell lines

Clinical specimens

EVs isolation and characterization

Flow cytometry

EVs analysis

HBEC analysis

Wound-healing assay

3D Proliferation assay

Statistical analysis

Data availability

References

Funding

Author information

Authors and Affiliations

Contributions

Corresponding author

Ethics declarations

Competing interests

Ethics

Additional information

Supplementary information

Supplementary materials

Reproducibility Checklist

Rights and permissions

About this article

Cite this article

Share this article

Search

Navigation

HIF-1α is transferred from COPD-EVs to HBEC-KRAS^V12high

HIF-1α shuttle induces CXCR4 modulation and CD133⁺ stem cells