7SK-enriched exosomes suppress the cancer phenotypes in human lung cancer cells: insights from 2D and microfluidic 3D in vitro models.

Introduction
Lung cancer remains the leading cause of cancer-related mortality worldwide, with almost 1.8 million deaths and around 2.5 million new cases in 20221. Despite therapeutic advances, clinical outcomes remain poor due to drug resistance, tumor heterogeneity, limited efficacy of targeted therapies, and toxicity of chemo- and radiotherapy2–4. Therefore, novel therapeutic approaches are urgently needed.
RN7SK (7SK) is a long non-coding RNA (lncRNA) that interacts with several proteins, such as MePCE, HEXIM1/2, and Larp7, to regulate gene transcription and thereby control essential cellular activities. This regulatory role is exerted by either the inhibition of RNA Polymerase II activity, primarily by suppressing P-TEFb kinase activity, or through the inhibition of HMGA1 activity5–9.
Despite extensive evidence highlighting the role of the aforementioned proteins in cancer progression9–14, the involvement of 7SK in cancer remains largely underexplored. In this context, our research team reported 7SK downregulation in breast, colon, and myeloid cancers15. Similarly, in tongue squamous cell carcinoma, reduced 7SK correlated with larger tumors, while its knockdown enhanced cell survival, migration, and tumorigenesis16. Recently, our team demonstrated that treatment of triple-negative breast cancer (TNBC) cells with 7SK resulted in suppression of some cancer phenotypes17.
The potential anticancer effects of 7SK on lung cancer, though indirectly supported by several pieces of evidence about the roles of 7SK-interacting proteins in cancer18–21, remain unknown and require further investigation. HMGA1, identified as one of the key partners of 7SK in transcription regulation (where its activity or expression is inhibited by 7SK)9,17, plays a recognized role in the onset and progression of lung cancer19. Some studies have demonstrated that elevated HMGA1 expression is commonly observed in lung cancer, correlating inversely with patient prognosis and survival18,19. In addition, HMGA1 upregulation directly contributes to the progression of lung cancer20,21. Moreover, m6A demethylation of 7SK has been reported to reduce colony formation of non-small-cell lung cancer (NSCLC) cells22. However, a recent study presented contrasting evidence, suggesting that 7SK may promote lung cancer. Elevated 7SK expression was observed in lung adenocarcinoma (LUAD) tumors compared to adjacent tissues. Additionally, 7SK knockout reduced the spheroid formation by lung cancer cell lines23. These evidences highlight the need to explore the hypothetical roles of 7SK against lung cancer.
Exosomes represent a physiologically relevant mode of intercellular RNA transfer and closely mimic endogenous mechanisms of RNA delivery within the tumor microenvironment. Compared with conventional overexpression approaches (e.g., plasmid or viral transfection), exosome-mediated delivery offers several advantages, including reduced cytotoxicity, avoidance of genomic integration, improved RNA stability, and the ability to deliver functional RNA in a transient yet biologically meaningful manner24–27. In addition, conventional overexpression systems, particularly plasmid- or viral vector–based approaches, offer limited control over the dosage and temporal dynamics of RNA expression once delivered into recipient cells28,29. Overall, the mentioned characteristic profile, combining accessibility, biocompatibility, and therapeutic potential, makes human umbilical cord-derived mesenchymal stem cell (hUC-MSC)-derived exosomes an ideal choice for advancing exosome-based treatments in cancer research.
Given evidence supporting 7SK’s anticancer role, we hypothesized that 7SK-loaded exosomes could suppress NSCLC phenotypes. We examined their effects on cell viability, apoptosis, tumorigenicity, and migration/invasion in 2D cultures and 3D spheroids within a microfluidic system mimicking in vivo conditions.

Methods

Cell culture
A549 cells were purchased from Bonyakhteh Stem Cell Bank, Stem cell technology research center: STRC (Tehran, Iran) and cultured in DMEM (Bio-Idea, Iran) enriched with 10% fetal bovine serum (FBS; Bio-Idea, Iran) and 1% penicillin-streptomycin (Pen-Strep, Bio-Idea, Iran) at 37 °C and 5% CO2. The culture media was changed every 2 days. Once the cells were 80% confluent, they were subcultured.

Approach for human umbilical cord-derived mesenchymal stem cell (hUC-MSC) isolation
Human umbilical cord tissue, obtained as biological waste following full-term delivery, was purchased from ROYAN Stem Cell Technology (Tehran, Iran). All samples were collected with informed parental consent and under institutional ethical approval, in compliance with national bioethics regulations. The human umbilical cord is processed under sterile conditions30. Briefly, vessels were removed, tissue fragments (3–5 mm) were digested with 0.25% trypsin-EDTA at 37 °C for 30 min, and cultured in DMEM/F12. After 2–3 weeks, adherent hUC-MSCs were harvested and passaged at ~ 80% confluency.

HUC-MSCs characterization
An optical microscope was utilized to determine the morphology of hUC-MSCs. Also, Flow cytometry was used to detect MSC-specific markers (CD90+, CD105+, CD45−, and CD34−) and, as a result, determine the purity of MSCs. In short, primary monoclonal fluorescence-labeled antibodies (BD Biosciences, USA) were used to stain hUC-MSCs (1 × 106 cells, passage 3, in 100 µl PBS). Afterward, cells were incubated at 4 °C (30 min). Detection was performed using a flow cytometer, and data were analyzed using FlowJo v10 software (FlowJo LLC). Additionally, to explore the multipotent characteristics of hUC-MSCs, we used osteogenic and adipogenic differentiation assays17.

Isolation of exosomes
HUC-MSCs, in exosome-depleted medium, were incubated for 72 h to allow cells to release exosomes into the media. Then, the conditioned media was collected. The media was centrifuged (3000 rpm for 10 min) at 25 ℃ for the removal of cellular debris and dead cells. The supernatant was passed through a 0.22 μm filter to ensure the removal of remaining large particles and debris. After that Exocib kit (EXOCIB; Cibbiotech, Iran) was utilized for exosome isolation according to the manufacturer’s instructions.

Characterization of exosomes

Electron microscopy
Transmission electron microscopy (TEM, Zeiss 192 EM900) and scanning electron microscopy (SEM, KYKY-EM3200, China) were used to examine the morphology of isolated exosomes, following the protocols outlined in our prior study17. Briefly, the exosome solution was fixed with 2.5% glutaraldehyde for approximately 12 h and 2% osmium tetroxide for 30 min. Fixed exosomes were dehydrated through an ethanol series. Subsequently, 812 Resin kit (TAAB, UK) was used. The sample staining procedure was then conducted. Images were captured by a TEM. For SEM imaging, exosomes were placed on a glass slide and left to dry at 25 °C. Once dried, images were taken using SEM.

Dynamic light scattering
Exosome samples were first diluted in PBS (1:6 v/v) to an appropriate concentration to optimize detection. The DLS measurements were then performed to determine the average particle size distribution, providing an accurate assessment of the exosome diameter.

Quantification of exosome proteins
The protein content of the exosomes was quantified utilizing the Bicinchoninic Acid (BCA) assay Kit (Ariatous, Mashad, Iran). Briefly, BCA working reagent was made. Then, 25 µl of either bovine serum albumin (BSA) or exosome sample was combined with 75 µl of BCA working reagent in a 96-well plate. The mixture was completely blended and incubated at 37 °C for 30 min to allow the colorimetric reaction to occur. Next, the absorbance was measured at 562 nm by an ELISA reader system (BioTek, USA).

In vitro synthesis of 7SK
For 7SK synthesis, we initially amplified the 7SK DNA sequence through PCR, using a pCDH-H1/7SK vector17. The DNA quantity was assessed with a NanoDrop spectrophotometer (Aosheng, China), and 1 µg of the 7SK DNA was then used for RNA synthesis. The reaction mixture included 6 µl of 10× buffer, 1.2 µl of 25 mM rNTP mix (BioLabs, USA), and 2 µl of RNase inhibitor (40 u/µl; GeneAll, Korea). After adding 1.5 µl of T7-RNA polymerase (BioLabs, USA), the reaction was incubated (37 °C for 4 h).

Exosome internalization assay
Exosomes were labeled utilizing PKH26 Red Fluorescent Cell Linker Midi Kit (Sigma-Aldrich, USA) based on the instructions provided by the manufacturer, with slight modifications. Briefly, 100 mg of exosomes were suspended in 500 µl of Diluent C. An equal amount of diluted PKH26 dye solution was then mixed with the exosome suspension and incubated at 37 °C for 5 min. The labeled exosomes were then isolated from any unbound dye by using the EXOCIB exosome precipitation reagent. The PKH26-stained exosomes were added to nearly confluent A549 cultured cells. Following a 4-hour incubation, cells were labeled with 1 µg/ml Hoechst 33342 (Thermo Fisher Scientific, USA) and incubated for 10 min at 37 °C. Then, after PBS rinsing, cells were fixed (4% paraformaldehyde) for 5 min at 25 °C, and subsequently, images were captured by a fluorescent microscope with a digital camera (TE2000-U, Nikon, Japan).

Delivery of RN7SK-loaded exosomes to A549 cells
Exosomes were suspended at a concentration of 0.5 µg/µl in a pre-cooled electroporation buffer. Subsequently, 7SK was added to the exosome suspension with a resulting concentration of 100 pmol 7SK per 100 µg/ml of exosomes. The solution was incubated on ice for 2 min and filled into a pre-refrigerated 4 mm electroporation cuvette. Electroporation was performed using an electroporator (Eppendorf, Germany) set to 400 V. Then, A549 cells were treated with 100 µg/ml of 7SK-loaded exosomes (Exo-7SK). Control groups consisted of untreated cells and cells subjected to unloaded exosomes (unloaded-Exo). The relative amount of 7SK in the treated A549 cells and their controls was quantified utilizing quantitative real-time PCR (qRT-PCR) after 24 h of treatment.

RNA isolation and qRT-PCR
RNA isolation procedure was performed through the RNA Extraction Kit (GeneAll, South Korea). Briefly, complementary DNA was synthesized from 1 µg of the purified RNA using the RevertAid M-MuLV Reverse Transcriptase Kit (Fermentase, Hanover, Germany). QRT-PCR was executed on a Step One machine through SYBR Green I Master Mix (Ampliqon, Denmark). The qRT-PCR results were analyzed through the 2−ΔΔCt calculation. Primer sequences for the reactions are presented in Supplementary Table 1.

MTT assay
A549 cells were plated in a 96-well plate at 104 cells per well and incubated at 37 °C with 5% humidified CO2. The following day, cells were exposed to Exo-7SK or corresponding controls. The culture medium was discarded at 24-, 48-, and 72-h post-treatment, and the cells were rinsed with PBS. Subsequently, a volume of 20 µl of MTT solution, at a concentration of 5 mg/ml in PBS, was introduced to each well, and the plate was incubated for 4 h. Following the incubation, the supernatant was discarded, and 100 µl of DMSO was introduced to each well. Cell viability was determined based on the absorbance values (measured at 570 nm) utilizing an ELISA reader.

Assessment of cell population and viability using trypan blue exclusion
5 × 105 A549 cells per well were seeded in a 6-well plate and incubated overnight in a complete culture medium to allow for attachment and growth. After that, the culture media was replaced with serum-free media, and the cells were subjected to Exo-7SK or appropriate controls and incubated for 24 h. The attached cells were harvested using trypsin, and their viability was assessed with 0.5% trypan blue dye solution.

Assessment of cell cycle progression
A549 cells were plated in 6-well plates at 1 × 105 cells per well and allowed to adhere overnight. Following 24 h of treatment, cells (Exo-7SK-treated or control groups) were harvested and fixated in 70% ethanol at 4 °C until the next day. After fixation, cells were rinsed twice with PBS and resuspended in 500 µl of PI staining solution (Sigma-Aldrich, Steinheim, Germany), consisting of 0.1 mg/ml RNase A, 0.1% Triton X-100 in PBS, and 50 µg/ml PI. The cell suspension was placed in the incubator for 30 min. The DNA content was assessed through flow cytometry. Data were analyzed using FlowJo software.

Caspase 3/7 activity assay
A549 cells were plated at 1 × 104 cells per 100 µl in 96-well culture plates and incubated at 37 °C. 5 µM Caspase-3/7 Green Detection Reagent (Thermo Fisher Scientific, USA) was added to the treated and control cells, and placed in an incubator for 30 min. 20 min later, cells were labeled with 1 µg/ml Hoechst 33342 and incubated at 37 °C with 5% CO2. Pictures were taken by a fluorescence microscope and analyzed through Fiji/ImageJ software.

Evaluation of apoptosis using annexin V/PI staining
A549 cells were plated in 6-well plates at 1 × 105 cells per well. After treatment, cells were trypsinized and harvested. Next, the Annexin V/PI staining was performed, and the obtained data through flow cytometry were analyzed17.

Wound healing assay
A549 cells were plated into 6-well plates at 5 × 105 cells per well and permitted to grow to nearly 100% confluence in a complete growth medium. A uniform scratch was induced in the cell monolayer utilizing a sterile 200 µl pipette tip. Fresh serum-free media was then added to wells, and the cells were treated with Exo-7SK or its controls. The plates were placed in the incubator. The wound area images were taken at 0 and 24 h using a phase-contrast microscope. The wound area was analyzed using Fiji/ImageJ software.

Transwell-based migration and invasion analysis
A transwell migration assay was conducted using 24-well plates with 8.0 μm pore inserts (SPL, Korea). A549 cells were resuspended in serum-free medium (2 × 105 cells/ml), and 200 µl of suspension (4 × 104 cells) was added to the upper chamber. After 3 h, cells were treated with Exo-7SK or control solutions, while the lower chambers received 600 µl of complete medium as a chemoattractant. Following 24 h of incubation, non-migrated cells were removed with a cotton swab. Migrated cells on the lower surface were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and rinsed with PBS.
For invasion analysis, transwell inserts were pre-coated with 50 µl of Matrigel (Corning, USA) diluted 1:8 in serum-free medium and polymerized at 37 °C for 1 h. Cell seeding, treatment, and staining followed the migration protocol. Migrated and invaded cells were counted under an inverted light microscope (Olympus, Japan) in five random fields per insert to calculate average cell counts.

Spheroid formation assay
Spheroids were formed in microwells with a diameter of 400 μm. 4 × 105 cells were seeded into each well, with about 1 ml of working medium. The plate was centrifuged at 1200 rpm for 3 min to settle the cells into the microwells. An additional 2 mL of medium containing Exo-7SK or the corresponding controls was introduced to each well. After 3 days, images were captured, and the size of the spheroids was subsequently measured.

Microfluidic 3D culture, and spheroid dispersion assay
To achieve spheroids of 40–100 μm in size, synthesized spheroids were sieved using 100 μm and 40 μm filters. After that, the resulting spheroid pellet was redispersed in collagen hydrogels (rat tail collagen type I, 3.36 mg/ml, Corning Co.), prepared at 2 mg/ml and pH 7.4. The hydrogels, embedded with spheroids, were introduced to the central channels of the microfluidic device (AIM BIOTECH, https://www.aimbiotech.com)31 and incubated for 30 min at 37 °C in a humidified chamber. After incubation, the hydrogels were rehydrated with complete DMEM and further incubated at 37 °C. For each group (group 1: untreated, group 2: Exo treated, Group 3: Exo-7SK treated), three devices were utilized to provide triplicate experimental conditions and were observed under a microscope every 24 h over a period of six days. Also, to investigate exosome internalization in an in vivo-like tumor environment, PKH26-stained exosomes were used, and images were captured using a fluorescent microscope.

Assessment of spheroid viability using AO/PI staining
To assess the viability of spheroids following various treatments, a Live/Dead assay was performed using the acridine orange (AO)/PI staining solution (Nexcelom, CS2-0106). The culture media was completely discarded from the side channels. 20 µl of the prepared staining solution was added to each well. The device was incubated at room temperature in the dark for 20 min. After incubation, the spheroids were examined under a fluorescence microscope to assess their viability. Images were captured to document the red and green fluorescence, indicating dead and live cells, respectively. The images were analyzed using Fiji/ImageJ software to quantify the total area of live cells (AO-positive) to dead cells (PI-positive).

Statistical analysis
Statistical analyses were conducted using GraphPad Prism version 9. Data are presented as the mean ± standard deviation (SD) from three independent assays. One-way ANOVA) and Tukey’s post-hoc test) was used for the assessment of significant differences between study groups.

Results

The isolated HUC-MSCs and HUC-MSC-derived exosomes showed standard characteristics
hUC-MSCs were characterized by morphology, immunophenotype, and multipotency. They displayed a spindle-shaped morphology (Fig. 1A) and expressed CD90 (85.7%) and CD105 (88.8%) but low CD34 (2.3%) and CD45 (0.45%), confirming mesenchymal identity (Fig. 1B). Adipogenic and osteogenic differentiation, verified by Oil Red O and Alizarin Red S staining, demonstrated multipotency (Fig. 1C,D).

Exosomes showed spherical morphology with intact membranes under SEM and TEM (Fig. 2A,B), and DLS analysis indicated an average diameter of 88.07 nm (Fig. 2C), confirming successful isolation and characterization.

Uptake of 7SK-enriched exosomes enhanced the 7SK levels in NSCLC cells
The uptake of exosomes by NSCLC cells and their 7SK levels after treatment with 7SK-loaded exosomes was confirmed using fluorescence microscopy and qRT-PCR. A549 cells were treated with labeled hUC-MSC-derived exosomes. Following a 24-h incubation period, most cells exhibited fluorescent signals, indicating successful uptake of exosomes (Fig. 3A). Interestingly, the same results were also achieved in the 3D microfluidic culture (Fig. 3B). This finding showed that exosomes can penetrate collagen gel and enter spheroids. Additionally, the 7SK level in A549 cells was significantly elevated, as determined by qRT-PCR, at 24 h post-treatment with Exo-7SK when compared to the controls (Fig. 3C). These findings validated the effective 7SK delivery to NSCLC cells via exosomes post-loaded with 7SK.

Treatment with 7SK-enriched exosomes suppressed NSCLC cell viability
The impact of Exo-7SK on the NSCLC cell viability was assessed through MTT assays conducted at 24, 48, and 72 h post-treatment. A significant reduction in the viability of A549 cells treated with Exo-7SK was observed at all examined time points as compared to the controls (Fig. 4A). Given that no significant difference was observed between the unloaded exosomes and the untreated group at the 24-h time point, in contrast to the 48- and 72-h time points, and to minimize the anticancer effects of exosomes in our experiments, the 24-hour treatment was chosen for subsequent analyses. Furthermore, our findings revealed that, unlike the 48- and 72-h time points, Exo-7SK treatment at the 24-h time point did not result in a statistically significant reduction in the viability of normal MRC-5 cells compared to the control groups (Fig. S1).

To further substantiate these findings, the Annexin V/PI assay was employed, showing a marked elevation in early- and late-stage apoptotic cells post-Exo-7SK treatment relative to controls (Fig. 4B,C). Exo-7SK-treated A549 cells exhibited a notable down-regulation of BCL-2 (an anti-apoptotic gene) and up-regulation of BAK1 and p53 (pro-apoptotic genes) compared to the control groups (Fig. 4D). Moreover, Exo-7SK activated caspase 3/7 in A549 cells, leading to a higher rate of apoptotic cells as compared to controls (Fig. 5A,B). These results may imply that the exosome-based 7SK delivery may significantly impair the viability of NSCLC cells.

Treatment with 7SK-enriched exosomes suppressed NSCLC cell proliferation
To assess the effect of Exo-7SK on A549 cell proliferation, cell cycle, and growth curve analyses were conducted. Upon treating the cells with Exo-7SK, a significant reduction in total cell counts and viability was observed when compared to the control groups (Fig. 6A,B). Following treatment with Exo-7SK, cell cycle analysis further revealed a statistically marked reduction in the percentage of cells in the S and G2/M phase in comparison to the controls (Fig. 6C,D). These findings suggest that the 7SK delivery via exosomes can effectively inhibit the proliferation of A549 cells, potentially through modulation of the cell cycle.

Treatment with 7SK-enriched exosomes inhibited NSCLC cell migration and invasion
To determine whether Exo-7SK can suppress the metastatic characteristics of A549 cells, transwell migration/invasion, and wound healing assays were performed, along with an assessment of genes involved in EMT. As depicted in Fig. 7A (wound healing assay), a noticeable accumulation of cells in the denuded zone was observed after 24 h in the control groups. However, A549 cells exposed to Exo-7SK revealed a reduced ability to migrate and fill the wounded area (Fig. 7A,B). The transwell assays revealed that treatment with Exo-7SK significantly reduced both the migration (Fig. 7C,D) and invasion (Fig. 7E,F) capabilities of A549 cells in comparison with the control groups. The results showed a significant rise in the expression of the EMT-suppressor gene E-cadherin and a marked decrease in the expression of EMT-promoter gene N-cadherin in the Exo-7SK group compared to the controls (Fig. 7G). Based on these findings, Exo-7SK effectively reduces the migratory and invasive capacity of A549 cells, potentially through modulation of EMT-related gene expression.

Treatment with 7SK-enriched exosomes decreased the size of NSCLC spheroids
To assess the effect of Exo-7SK on the tumor formation ability of NSCLC cells, A549 cells underwent a spheroid formation assay. After treating the cells with Exo-7SK, a significant reduction in the size of spheroids was observed when compared to the control groups (Fig. 8A,B).

Treatment with 7SK-enriched exosomes inhibited the dispersion of NSCLC spheroids in 3D microfluidic culture
To assess the former results in a more advanced in vitro tumor model highly recapitulating the in vivo conditions, a microfluidic 3D lung cancer model was utilized (Fig. 9A). NSCLC spheroids derived from A549 cells were embedded within a hydrogel matrix and injected into the central channel of a microfluidic device. Subsequently, a culture medium containing Exo-7SK was introduced into the side channels (Fig. 9A). This model enabled a spheroid dispersion assay, which represents the EMT as the initial/crucial step in metastasis31. Treatment with Exo-7SK significantly suppressed spheroid dispersion, reducing the total dispersion area compared to the control groups (Fig. 9B,C). Without Exo-7SK treatment, a substantial dispersion of A549 spheroids cultured on the central channel was observed. These findings demonstrate that Exo-7SK may play a role in inhibiting EMT and metastasis in an in vivo-like tumor microenvironment.

Treatment with 7SK-enriched exosomes reduced the viability of NSCLC spheroids in 3D microfluidic culture
To further assess the effects of Exo-7SK on the growth of A549 cells in the 3D-microfluidic model, a live/dead assay using AO/PI staining was performed. After 6 days of incubation, the dead cell percentage in non-treated and exosome-treated spheroids was significantly lower compared to live cells. In contrast, Exo-7SK-treated spheroids exhibited an increased rate of dead cells (Fig. 10A,B). Overall, these data further indicate that 7SK delivery may reduce the survival/growth of A549 cells.

Discussion
7SK is a lncRNA showing regulatory roles in transcription through interaction with multiple proteins within a ribonucleoprotein complex5. Limited findings support the hypothesis that 7SK may play a role in suppression of the cancer phenotypes15–17. Moreover, the generalizability of these findings to other types of cancer needs further investigation. In this study, we tested the hypothetical anticancer effects of 7SK against NSCLC, which poses a huge burden on global health2. Our findings, using 7SK-loaded exosomes to increase 7SK levels in NSCLC cells, provided partial evidence for this hypothesis. 2D experiments demonstrated that exosome-based 7SK delivery significantly reduced the viability, proliferation, migration, and invasion in NSCLC cells. Furthermore, in a 3D microfluidic lung cancer model, 7SK delivery markedly inhibited spheroid formation ability and dispersion of tumor spheroids, and induced cancer cell death, confirming its anti-metastatic and anti-proliferative properties. Collectively, these findings suggest that 7SK may have anticancer effects on NSCLC.
Here, we observed anticancer phenotypes upon 7SK delivery, consistent with our previous report in TNBC cells17. Our findings suggest that the potential anticancer effects of 7SK delivery extend beyond TNBC and could be leveraged to suppress NSCLC. Our results support earlier reports on the progression of cancer phenotypes in TSSC following 7SK inhibition and the downregulation of 7SK in cancerous tissues and cells16.
This study provides preliminary evidence that delivery of unmethylated 7SK inhibits the cancer phenotypes in NSCLC cells. However, our findings might appear contradictory to a previous study by Xu et al., which showed that 7SK is a target of m6A methylation in lung cancer and, thereby, considering the known role of m6A methylation in tumorigenesis, suggested 7SK as a cancer-promoting factor23. In particular, they reported an impairment of spheroid formation in A549 cells upon 7SK knockout. In addition, they reported higher levels of 7SK in LUAD samples compared with adjacent normal tissues, as well as a negative correlation between 7SK levels and survival of LUAD patients23. These divergences might have stemmed mainly from the distinct nature of the unmethylated 7SK used in our study, which had been originally intact in terms of m6A methylation. Once delivered into cells, this form of 7SK may shift the intracellular balance in favor of unmethylated 7SK and adopt a conformation that enhances P-TEFb sequestration, a process that has been reported to inhibit RNA Pol II activity, transcription, and colony formation in NSCLC cells22.
The results of this study demonstrate that 7SK-enriched exosomes effectively elevate 7SK levels in NSCLC cells. This finding aligns with and extends our previous observations in TNBC cells17, reinforcing the broader potential of exosome-mediated 7SK delivery across cancer types. Although the use of exosomes for delivering small nucleic acid fragments has been extensively documented, reports on the delivery of lncRNAs via exosome post-loading are limited to our recent studies. We also demonstrated that exosomes can penetrate collagen hydrogels, traverse an in vivo-like tumor microenvironment, and enter spheroids. Exo-7SK treatment markedly inhibited spheroid dispersion, reducing migration compared to controls. In contrast, untreated A549 spheroids showed extensive spreading. Notably, this study represents the pioneering report of lncRNA delivery in a microfluidic cancer model. Given the growing therapeutic potential of lncRNAs and the relative safety and targetability of exosomes, these findings underscore the importance of exosomal delivery for future clinical studies based on lncRNAs.
In our MTT assay, hUC-MSC-derived exosomes showed marked anticancer activity at 48 and 72 h, unlike the 24-hour point used for further tests. This contrasts with our TNBC findings, where they had no significant effect on cancer-related phenotypes17, and with a report showing LUAD promotion32, but aligns with an NSCLC study showing tumor suppression33. Differences in isolation methods, MSC source, cancer cell line, and cancer model may explain these inconsistencies34–38.
This study provides preliminary evidence that delivery of 7SK may inhibit the cancer phenotypes in NSCLC cells. These findings are consistent with prior reports demonstrating that HMGA1 overexpression drives the progression of this cancer18–21. This alignment is supported by the earlier evidence on the inhibitory effects of 7SK against HMGA1 activity9 or expression17. While this insight is consistent with existing evidence, the exact molecular pathways through which 7SK modulates HMGA1 in NSCLC remain to be elucidated and represent an important direction for future studies.
Lack of a proper RNA control has been a limitation of our study, similar to other studies on lncRNAs. Such a limitation stems from the challenges of providing RNA controls for lncRNAs, considering their noncoding but regulatory nature, along with their long sequence and intricate secondary and tertiary structures. This limitation makes it difficult to draw a firm conclusion. However, moving forward to clinical studies, such a limitation needs to be addressed, especially to exclude the possible effects of immune response against in vitro transcribed RNAs.
While the present study provides valuable insights, several shortcomings remain that need to be examined in future studies. In particular, employing RNA immunoprecipitation (RIP) and co-immunoprecipitation (Co-IP) assays will be necessary to verify the incorporation of delivered 7SK into relevant regulatory complexes, 7SK/P-TEFb RNP or the 7SK/HMGA1 RNP, and to elucidate the precise molecular mechanisms underlying its anticancer activity. Expanding the investigation to additional cancer cell lines will help establish the broader applicability of these findings. Moreover, incorporating in vivo studies using clinically relevant tumor models will further enhance the translational potential of exosome-mediated 7SK delivery. Future work may also benefit from employing advanced systems such as patient-derived organotypic tumor spheroids that capture critical processes like angiogenesis and metastasis. Together, these efforts will strengthen the evidence base and support the continued development of this innovative therapeutic strategy against lung cancer.

Conclusion
Here, we provided partial evidence that exosome-based 7SK delivery may suppress NSCLC cell viability, proliferation, migration, and invasion in both 2D and 3D culture models. These findings highlight the therapeutic potential of 7SK delivery via exosomes as a novel strategy for NSCLC treatment. However, such conclusions require confirmation through further comprehensive studies examining both phenotypic and mechanistic specificity.

Supplementary Information
Below is the link to the electronic supplementary material.