Anaplastic lymphoma kinase-positive (ALK⁺) anaplastic large cell lymphoma (ALCL) is an aggressive peripheral T-cell lymphoma representing 10%-15% of pediatric lymphoid neoplasms. The constitutively activated nucleophosmin-ALK (NPM-ALK) fusion protein is the main driver of ALK⁺ ALCL oncogenesis [1]. Current clinical protocols can achieve high cure rates, with 75% event-free survival (EFS) at 5 years [2], but the patients’ outcome with relapsed/refractory disease still remains poor [3]. Liquid biopsy is widely applied for the prognostic and therapeutic stratification of onco-hematological patients. As a non-invasive procedure, it can be used to identify several biomarkers, such as small extracellular vesicles (S-EVs) [4]. S-EVs are stable carriers of molecules that mediate genetic exchange between distant cells, alter the microenvironment, facilitate cancer cell progression and promote drug resistance [5].

To investigate the role of S-EVs-delivered microRNAs (miRNAs) as disease biomarkers and their function in pediatric ALCL, we identified plasma S-EVs miRNA profile by small RNA-sequencing (sRNA-seq) in a panel of NPM-ALK⁺ ALCL patients (n = 20) and healthy donors (HD) (n = 5). All ALCL patients included in the study were NPM-ALK⁺; henceforth, we will refer to them as “ALCL”. The patients’ baseline characteristics are shown in Supplementary Table S1. Bioinformatics analysis using the miR&moRe2 software [6] showed distinct miRNA profiles between ALCL and HD (Figure 1A) with 12 miRNAs significantly enriched in ALCL S-EVs (Supplementary Figure S1A and Supplementary Table S2). For further investigations, we focused on miR-122-5p since it is involved in the dissemination of adult solid tumors [7]. miR-122-5p enrichment in plasma S-EVs of an extended cohort of ALCL (n = 66) patients and HD (n = 19) was validated (Figure 1B), and the highest levels were detected in patients with disease dissemination (stage III-IV) (Figure 1B). Of note, comparing miR-122-5p levels in ALCL plasma S-EVs with patient-matched tumor biopsies, we found that miR-122-5p was barely detectable in ALCL lymph nodes according to both sRNA-seq (Supplementary Figure S1B) and real time-quantitative polymerase chain reaction (RT-qPCR) (Figure 1C) analyses. miR-122-5p, described as liver-derived, accounts for 52% of whole hepatic human miRNome [8]. We analyzed miR-122-5p expression in ALCL biopsies from different tissues and confirmed that miR-122-5p was almost or completely absent in all samples except the liver (Supplementary Figure S1C). Moreover, in a panel of ALCL cell lines, miR-122-5p was not expressed, while alpha mouse liver 12 (AML12) cells and S-EVs showed high miR-122-5p levels (Supplementary Figure S1D). These results strongly suggest that miR-122-5p circulating in plasma S-EVs of ALCL patients is not released by the tumor cells but most likely derives from the liver. In line with these observations, we demonstrated the presence of miR-122-5p only in the non-tumoral CD30⁻ S-EVs fraction (Supplementary Figure S1E). Interestingly, we observed significantly higher transaminase values at diagnosis in ALCL patients with miR-122-5p levels above the median value (Supplementary Figure S1F). Therefore, we hypothesized that the increased circulating levels of miR-122-5p might result from disease-related liver function impairment in patients with advanced ALCL.

To elucidate the role of miR-122-5p in disease dissemination and aggressiveness, we conducted a sequence-based miRNA target prediction with TargetScan (http://www.targetscan.org/). Our results showed that pyruvate metabolism and glycolysis were the most enriched pathways that involved miR-122-5p targets (Supplementary Figure S2). miR-122-5p validated targets are citrate synthase (CS), pyruvate kinase M2 (PKM2) [7] and aldolase A (ALDO A) [9]. We hypothesized that liver-derived S-EVs enriched in miR-122-5p could sustain ALCL dissemination by inhibiting glycolysis in niche cells, thus increasing glucose availability to support cancer cell seeding (Figure 1D). When we treated mouse embryonic fibroblast-1 (MEF-1) as a niche cell model with AML12-derived S-EVs (S-EVs isolation in Supplementary Figure S3), we observed that MEF-1 cells were able to efficiently uptake AML12-derived S-EVs (Figure 1E) and showed increased miR-122-5p intracellular levels compared to control (Figure 1F). Furthermore, S-EVs treatment significantly inhibited the expression of ALDO A (Figure 1G). Moreover, glucose transporter 1 (GLUT1) expression was significantly decreased in cells treated with S-EVs compared to the untreated (NC) group (Figure 1G). We transiently transfected MEF-1 cells not expressing endogenous miR-122-5p with miR-122-5p-mimic (Figure 1H). Significant downregulation of PKM2, CS and ALDO A transcript levels (Figure 1I) was detected in miR-122-5p overexpressing cells at 24, 48 and 72 hours post-transfection. A slight decrease of all miR-122-5p analyzed targets protein was appreciated 24 hours post-transfection (Figure 1J). Moreover, GLUT1 transcript expression was significantly decreased 24 hours post-transfection (Figure 1I), and glucose concentration was increased in MEF-1 cells media overexpressing miR-122-5p (Figure 1K), suggesting that miR-122-5p impacts also on glucose uptake. Moreover, miR-122-5p transfected MEF-1 cells showed impaired glucose consumption, decreased extracellular acidification rate (ECAR) and a significantly lower glycolytic capacity than controls after 24 hours posttransfection (Figure 1L). The results obtained with HEK-293T cells stably overexpressing miR-122-5p (HEK-293T_miR-122-5p) were consistent with those of MEF-1 cells (Supplementary Figure S4). ALCL cells viability was significantly increased upon treatment with conditioned medium (CM) derived from HEK-293T_miR-122-5p (CM_miR-122-5p) compared to the control HEK-293T_mock (CM_mock) (Figure 1M and Supplementary Figure S5A). Also, the treatment with CM from HEK-293T_miR-122-5p transfected with miR-122-5p inhibitor reverted the effect of miR-122-5p on ALCL cell glycolysis and viability (Supplementary Figure S6). Interestingly, CM from MEF-1 cells treated with hepatocyte-derived S-EVs increased ALCL cells’ viability (Supplementary Figure S7). Similarly, both the invading and clonogenic potential of ALCL cells significantly increased after treatment with CM_miR-122-5p compared to CM_mock (Figure 1N-O and Supplementary Figure S5B-C). To analyze whether the effect of miR-122-5p contributed to the metabolic rewiring of ALCL cells, we analyzed the ECAR of tumor cells treated with CM_miR-122-5p or CM_mock. ALCL cells showed higher levels of ECAR and glycolytic capacity when treated with CM_miR-122-5p compared with CM_mock (Figure 1P and Supplementary Figure S5D). Overall, these results indicate that miR-122-5p impaired niche cells’ glucose utilization, favoring ALCL cells’ growth and aggressiveness.

To clarify in vivo the role of miR-122-5p in promoting premetastatic niche formation, mice were subjected to multiple treatments with miR-122-5p-enriched S-EVs (S-EVs miR-122-5p) or negative control S-EVs (S-EVs mock) three times a week for three weeks, GFP-labeled Karpas 299 cells were then injected (Supplementary Figure S8) and, after 20 days, the mice were sacrificed, and their lungs, lymph nodes, livers and spleens were collected and analyzed to assess the tumor cell spread. The presence of tumor cells was determined by flow cytometry. We found a significantly increased number of GFP-positive cells in the lungs of mice treated with S-EVs miR-122-5p compared to S-EVs mock (Figure 1Q). ALK staining confirmed the tumor cell spread in the lungs of mice treated with S-EVs miR-122-5p compared with S-EVs mock (Figure 1R and Supplementary Figure S9). Moreover, we found significantly increased tumor cell-specific NPM-ALK transcript levels in the lungs and lymph nodes of mice treated with S-EVs miR-122-5p compared to the S-EVs mock (Figure 1S-T).

In conclusion, we described the miRNA profile of plasma S-EVs from pediatric ALCL patients highlighting the role of miR-122-5p in promoting tumor cell dissemination and aggressiveness, despite its non-tumoral origin. This work underlines that ALCL represents a highly heterogeneous ecosystem where malignant and stromal cells collaborate to sustain tumor aggressiveness. Noteworthily, miR-122-5p antagonists, currently in clinical trials [10], could represent an effective therapeutic target for ALCL pediatric patients with high disease dissemination.

DECLARATIONS

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

This study was approved by the ethics committee of each participating institution (CESC n.4256/AO/17). Written informed consent was obtained from parents or legal guardians before patients’ enrollment.

The in vivo study was approved by the Local Ethical Committee and received Ministerial Authorization (n°788/2018-PR released on 10/15/2018).

CONSENT FOR PUBLICATION

Not Applicable