Midkine promotes tumor growth and attenuates the effect of cisplatin in small cell lung cancer

doi:10.21203/rs.3.rs-4918939/v1

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Midkine promotes tumor growth and attenuates the effect of cisplatin in small cell lung cancer

https://doi.org/10.21203/rs.3.rs-4918939/v1

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Purpose

Small cell lung cancer (SCLC) is a highly aggressive disease with poor survival. Nevertheless, the addition of an anti-programmed death ligand 1 antibody to platinum combination chemotherapy can improve its prognosis. However, only a few patients achieve a long-term response; thus, establishing new therapies for SCLC is crucial. Midkine (MDK) is a heparin-binding growth factor that plays a role in various biological processes such as cell proliferation and chemotherapy resistance in diverse cancers. MDK has garnered attention as a therapeutic and diagnostic target for several cancers; however, studies evaluating its expression and function in SCLC are scarce.

Methods

The MDK expression was analyzed in vitro and in vivo by ELISA, immunohistochemistry, western blotting. The effect of MDK on cell proliferation and the effect of cisplatin was evaluated by MTT assay.

Results

MDK was expressed pathologically in human SCLC tumor tissues but not in normal lung tissues. Human serum MDK concentration in patients with SCLC reflected the SCLC tumor burden and was correlated to the response to treatment. Moreover, MDK induced cell proliferation and attenuated the effect of cisplatin in SCLC cell lines. The combination of an MDK inhibitor and cisplatin exerted synergistic antitumor effects both in vitro and in vivo. Additionally, MDK positively regulated the AKT pathway.

Conclusion

The present results indicate that MDK contributes to cell proliferation and chemotherapy resistance by activating the AKT pathway in SCLC. Therefore, MDK may be a potential therapeutic and diagnostic target for SCLC.

midkine

small cell lung cancer

AKT

tumor progression

biomarker

therapeutic target

Small cell lung cancer (SCLC), accounting for approximately 14% of all lung cancers, is an aggressive disease, with most patients presenting metastatic stages at diagnosis, which further correlates to a poor survival rate [1]. Platinum-based chemotherapy is the standard of care in extensive-stage SCLC, eliciting responses in most cases; however, patients have been reported to rapidly develop therapy resistance [2]. Reportedly, adding an anti-programmed death ligand 1 antibody to the platinum combination chemotherapy can improve median survival; however, the effective long-term response has been reported in only a few patients [3, 4].

SCLC subtypes have recently been characterized and defined based on the relative expression of genes, namely ASCL1, NEUROD1, POU2F3, and YAP1, which encode four major transcriptional regulators. While SCLC with upregulated achaete-scute family bHLH transcription factor 1 (ASCL1) or neuronal differentiation 1 (NEUROD1) expression is a classic type with a neuroendocrine (NE) feature, other SCLC subtypes lack NE features, making SCLC a highly heterogeneous tumor [5–9]. The neurogenic locus notch homolog protein (Notch) pathway and Myc play important roles in SCLC heterogeneity [10, 11]. Although each subtype exhibits specific drug vulnerabilities [12–15], targeted therapies for SCLC have yet to be established. Therefore, it is imperative to establish targeted therapies for SCLC.

Midkine (MDK), a heparin-binding growth factor, was first discovered as a highly expressed gene during mouse embryogenesis [16]. MDK is a multifunctional secreted protein that interacts with various plasma membrane receptors [17]. MDK is highly expressed in various malignancies, and it contributes to cell proliferation, evasion from apoptosis, and rewiring the tumor microenvironment (TME) toward an immune-resistant state by activating intracellular pathways [17–23]. Therefore, MDK has been focused on as a therapeutic target [24, 25]. In SCLC, MDK, which is produced by non-NE SCLC cells, promotes the survival of NE SCLC cells [10]. Moreover, MDK is associated with resistance to cisplatin (CDDP) and immune checkpoint inhibitors (ICI) and is a potentially promising therapeutic target for SCLC [22, 23, 26, 27]. However, studies evaluating the expression and function of MDK in SCLC are scarce. Therefore, herein, we investigated the expression and function of MDK in SCLC.

2.1 Reagents

An MDK inhibitor (iMDK) was purchased from Merck (508052, Merck, Darmstadt, Germany), which was dissolved in DMSO and stored at − 30°C until further use. Recombinant human MDK (rhMDK) was purchased (450 − 16, Peprotech, RockyHill, NJ, USA), dissolved in nuclear-free water at 100 ng/mL, and stored at − 80°C until further use. An AKT inhibitor (iAKT) was purchased from Selleck Biotech (MK2206, Selleck Biotech, Kanagawa, Japan), dissolved in DMSO, and then stored at − 80°C until further use.

2.2 Cell lines and establishment of CDDP-resistant lung cancer cell lines

We used nine SCLC cell lines in this study. SBC5, SBC3, MS1L, and RERF-LC-MA were procured from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan), whereas H592, H1688, H209, H82, and H69 from the American Type Culture Collection (Manassas, VA, USA). SBC5, SBC3, and RERF-LC-MA were maintained in the minimum essential medium, whereas MS1L, H1688, H592, H209, H82, and H69 were maintained in the RPMI 1640 medium supplemented with 10% FBS and 100 U/mL penicillin–streptomycin in a humidified atmosphere at 37°C with 5% CO₂. Because of quality control issues related to RERF-LC-MA cells, only subgroup markers, MDK expression, and Notch signaling were evaluated in conjunction with other cell lines. Based on a previous study, a CDDP-resistant SCLC cell line, SBC5R, was established from their parental cell lines, SBC5, respectively. The parental cell line was treated with gradually increasing CDDP concentrations (maximum 2 µmol/L), followed by culturing in a medium containing 2 µmol/L CDDP for 3 months.

2.3 ELISA for human MDK and tumor burden evaluation

The blood samples of patients with SCLC at Hokkaido University Hospital were collected before first-line chemotherapy, after two and four cycles of chemotherapy, and during tumor progression between April 2020 and December 2023. Histopathological diagnoses were made by pathologists at the Pathological Institute of Hokkaido University Hospital. Furthermore, blood samples were collected from 10 healthy controls (median age: 35.6) with no history of malignancy and smoking. Additionally, the conditioned medium from the SCLC cell lines was collected and stored with these blood samples at − 30°C until evaluation. Human serum MDK concentration (s-MDK) and MDK concentrations in the culture medium were measured using an ELISA kit for human MDK (ab193761; Abcam, Sydney, Australia) according to the manufacturer’s instructions. Optical density was measured at 450 nm using a microplate reader (Varioskan Flash; Thermo Fisher Scientific, Waltham, MA, USA). Based on the results, the receiver operating characteristic (ROC) curve was constructed, and the corresponding area under the curve (AUC) values were calculated. Using the ROC curve, an optimized cut-off value (53.7 pg/ml) was defined for s-MDK regarding sensitivity and specificity. In patients with SCLC, high- and low-MDK expression groups were defined using the above s-MDK cutoff value. Tumor volume was calculated as the baseline sum of the longest diameter (BSLD) of the target lesion [28]. BSLD was calculated by measuring the target lesions based on the Response Evaluation Criteria for Solid Tumors Version 1.1, and the median (BSLD: 80.12 mm) was used as a cutoff value to divide the patients into high- and low-BSLD groups.

2.4 Immunohistochemistry (IHC)

ASCL1, NEUROD1, POU class 2 homeobox 3, yes-associated protein 1, synaptophysin (SYP), RE1 silencing transcription factor (REST), and MDK expression in human tumor tissues from four patients with SCLC was analyzed by IHC. MDK expression in dissected xenograft tumors was analyzed by IHC. Next, 5-µm thick tumor tissue sections from paraffin-embedded blocks were subjected to IHC staining using the specific antibodies listed in Table S1.

2.5 Antibodies and western blotting

Whole-cell lysates were subjected to western blotting and analyzed for protein levels using the specific antibodies listed in Table S2. Band intensity was calculated by quantitative densitometric analysis using ImageJ ver. 1.53 a (National Institutes of Health, Bethesda, MD, USA). Standardization was performed by measuring actin levels in the same blots using an anti-actin antibody (1:1500; A2066, Sigma-Aldrich, St. Louis, MO, USA). The experiments were performed in triplicates.

2.6 Cell proliferation assay

Cell growth was assessed by performing the MTT cell proliferation assay according to the manufacturer’s instructions (Thermo Fisher Scientific), and light absorption at 560 nm was determined using a microplate reader. SCLC cells were seeded at a density of 1 × 10³/well onto 96-well plates containing rhMDK. shMDK-overexpressing cells were seeded at a density of 1 × 10³/well onto 96-well plates for 96 h, followed by the MTT assay. The cytotoxicity of each drug was assessed. Tumor cells were seeded at a density of 5–20 × 10³ (SBC5 and SBC3: 5 × 10³, MS1L and H82: 10 × 10³, and H69: 20 × 10³)/well onto 96-well plates for 24 h. The medium was replaced with a fresh medium containing each drug. The cells were then treated for 72 h, and IC₅₀ was calculated based on the results (Tables S3 and S4). Synergism between CDDP and iMDK was quantified by performing the combination index analysis adapted from the median–principle methods of Chou and Talalay [29] using CalcuSyn 2.1 (Biosoft, Bengaluru, India). The experiments were performed in triplicates.

2.7 Xenograft mouse model

SBC5 control and SBC5 small hairpin (sh) RNA cells (5 × 10⁶ cells) were diluted in 200 µL PBS and injected subcutaneously into the right posterior leg of athymic 5-week-old female nude (nu⁺/nu⁺) mice. The tumors were measured daily using digital calipers, and the tumor volume was calculated as width² × length/2 [30]. Drug treatments were initiated when the mean tumor volume reached approximately 150–250 mm³. Body weights and general conditions were monitored. CDDP was administered by intraperitoneal injection once a week (1 mg/kg/day), and iMDK was administered by intraperitoneal injection thrice a week (9 mg/kg/day) [25]. Each group consisted of four mice. Two independent experiments were performed.

2.8 Statistical analyses

All data were derived from at least three independent experiments and are presented as the mean ± standard deviation unless indicated otherwise. Differences between the groups were analyzed using Welch’s t-test and multiple comparison tests. Statistical significance was set at p < 0.05. Statistical analyses were performed using GraphPad Prism v.10.0 (GraphPad Software, Software, San Diego, CA, USA). Additional methods are detailed in the Supplemental Document S1.

3.1 s-MDK reflects SCLC tumor burden

We measured s-MDK levels in 45 treatment-naive patients with SCLC and 10 healthy controls by performing ELISA. The profiles of these patients are summarized in Table S5. No significant difference was observed in patient characteristics between the high- and low-MDK expression groups except for treatment. Patients with SCLC showed significantly higher s-MDK levels than did the healthy controls (p < 0.05) (Fig. 1a). Based on the ROC curve analysis, an AUC value of 0.81 was obtained (Fig. 1b). s-MDK levels increased with advanced stages (Fig. 1c) and increased significantly in patients with high BSLD (Fig. 1d). In the high-MDK expression group, s-MDK levels decreased significantly after tumor responses and increased during tumor progression (Fig. 1e and f). In a patient with markedly increased s-MDK levels after four cycles of chemotherapy, the tumor progressed immediately afterward even though no increases in the level of pro-gastrin-releasing peptide, a representative SCLC marker, was observed (Supplementary Fig. S1a). A trend toward better prognosis was observed in the low-MDK expression group than in the high-MDK expression group (Supplementary Fig. S1b and c).

3.2 MDK expression in SCLC is variable and has no clear correlation with subgroups or NE features

Next, we evaluated MDK expression in surgically resected human SCLC tumors. Notably, MDK was detected in the cytoplasm after staining, whereas it was not observed in normal lung tissues (Fig. 2a). Moreover, the expression of MDK, SCLC transcriptional subtype markers, the NE marker SYP, and the non-NE marker REST was evaluated by IHC using four surgical sections of human SCLC. Immunostaining images are shown in Supplementary Fig. S2. The upregulated and widespread expression of MDK was observed in Case 2, whereas in other cases, MDK was expressed in some parts of the tumor. No correlation was found between the expression of MDK and SCLC subtype markers or NE markers among the four cases. Next, we determined the protein and mRNA levels of MDK in nine human SCLC cell lines by western blotting and qRT-PCR, respectively (Fig. 2b and c). Furthermore, MDK secretion in the nine cell lines was measured by ELISA (Fig. 2d). MDK was most upregulated and secreted in SBC5. Its modest or weak expression and secretion were observed in the remaining cell lines except for MS1L (Fig. 2b–d). Further, SCLC subtype markers, SYP and REST expression in each cell line was evaluated by western blotting (Fig. 2b). No clear correlation was observed between MDK expression and SCLC subgroups or NE features (Fig. 2b, Supplementary Fig. S2).

3.3 MDK regulates cell proliferation and migration in SCLC

The analysis of the effect of MDK on proliferative and migratory capacity in SCLC showed that rhMDK administration to SCLC cells with no or low MDK expression promoted cell proliferation (Fig. 3a). Additionally, MDK overexpression using the MDK expression vector promoted cell proliferation in SBC3 and MS1L (Fig. 3b). In contrast, MDK suppression by shMDK in SBC5 impaired tumor cell proliferation (Fig. 3c). iMDK inhibited MDK expression (Fig. 3d) and exerted concentration-dependent cytotoxic effects on SCLC cells expressing MDK, but not on MS1L cells not expressing MDK (Fig. 3e, Table S3). Furthermore, suppression of MDK expression by iMDK and shMDK decreased cell migration capacity in SBC5 cells, whereas rhMDK administration and MDK overexpression improved this capacity in MS1L (Fig. 3f and g).

3.4 MDK regulates intracellular signaling pathways in SCLC

Because MDK activates the PI3K/AKT and MAPK pathways and contributes to cell survival [17], we evaluated if MDK affected these signaling pathways. Suppression of MDK expression by shMDK in SBC5 downregulated P-AKT expression. Moreover, iMDK downregulated P-AKT expression in three cell lines (Fig. 4a). Although shMDK did not alter P-PI3K expression in SBC5 cells, iMDK downregulated its expression in three cell lines (Fig. 4a). MDK overexpression in SBC3 and MS1L upregulated P-PI3K and P-AKT expression (Fig. 4b). Conversely, MDK suppression did not alter P-ERK expression in SBC5 and SBC3, whereas this suppression upregulated P-ERK expression in H69 (Fig. 4a). MDK overexpression did not change P-ERK expression in SBC3 cells but upregulated it in MS1L (Fig. 4b). Notably, iAKT administration inhibited cell proliferation of SBC5 and SBC3 (Fig. 4c and d). Additionally, iMDK notably induced apoptosis (Fig. 4e) and increased cleaved (c)-poly-ADP ribose polymerase and c-caspase 3 levels (Fig. 4f). The Notch pathway plays an important role in tumor heterogeneity in SCLC [11], and Notch2, an MDK receptor candidate, is an airway neuroendocrine stem cell marker [31]. Therefore, herein, the expression of Notch pathway-related proteins was evaluated in SCLC. All MDK-expressing SCLC cell lines except H209 and H69 were positive for Notch2 intracellular domain expression (Fig. 2b–d, Supplementary Fig. S3a). Furthermore, MDK knockdown in SBC5, the SCLC cell line with high MDK expression and Notch pathway activation, resulted in the suppression of the Notch pathway (Supplementary Fig. S3b). In contrast, overexpressing MDK in SBC3 and MS1L did not activate the Notch pathway, including Notch2 (Supplementary Fig. S3c). These results suggest that in some SCLC cells, MDK activity is associated with the Notch pathway and that Notch2 might be a candidate receptor for MDK.

3.5 MDK attenuated the antitumor effect of CDDP in SCLC

AKT pathway can induce resistance to CDDP [32]; hence, we investigated if MDK can affect the antitumor effect of CDDP in SCLC. Notably, shMDK-mediated MDK suppression in SBC5 enhanced the antitumor effects of CDDP (Fig. 5a, Table S4). Conversely, MDK overexpression in SBC3 and MS1L attenuated the effects of CDDP (Fig. 5b, Table S4). Additionally, the synergistic effects of CDDP and iMDK were observed in all SCLC cell lines (Fig. 5c). SBC5R (Fig. 5d, Table S3) exhibited upregulated MDK expression and stronger PI3K/AKT pathway activation than SBC5 (Fig. 5e). Herein, shMDK-mediated MDK suppression in SBC5R suppressed the AKT pathway and enhanced the antitumor effects of CDDP (Fig. 5f and g), along with impairing the tumor cell proliferation (Fig. 5h). These results suggest the involvement of MDK in CDDP resistance in SCLC.

3.6 Suppression of MDK inhibits tumor growth of SCLC and enhances the effect of CDDP in vivo

We examined if MDK suppression inhibited tumor growth in vivo. The tumor burden in nude mice implanted with shMDK SBC5 cells was significantly lower than that in the control mice (Fig. 6a). Decreased MDK protein levels were maintained in resected tumors with shMDK at day 28 after tumor induction (Fig. 6b). Nevertheless, the iMDK and CDDP combination inhibited tumor progression more strongly than did the monotherapy (Fig. 6c). No significant weight loss was observed during the treatment (Fig. 6d). Additionally, MDK expression was suppressed after iMDK administration with or without CDDP in the resected tumors (Fig. 6e).

Herein, we demonstrated that MDK serum levels increased in patients with SCLC, and MDK regulated cell proliferation and the antitumor effect of CDDP in vitro and in vivo. To the best of our knowledge, this is the first study to evaluate the expression and functions of MDK in SCLC.

Several clinical trials on targeted therapies for SCLC have been performed and numerous are ongoing; however, the reported data are unsatisfactory [33]. SCLC is heterogeneous, and various SCLC subtypes exist simultaneously within a tumor, indicating that comprehensive treatment, and not just a single treatment, is necessary for the cancer [5, 10, 11, 34]. Lim et al. proposed that non-NE-type SCLC cells secrete MDK, which promotes the growth of NE-type SCLC cells [10]. Thus, we hypothesized that MDK-targeted therapy might be sufficient for treating tumors with various SCLC cells because MDK might target non-NE-type SCLC cells, thereby indirectly inhibiting the proliferation of NE-type SCLC cells. Although the present results did not show a correlation between SCLC subtypes or NE features and MDK expression in human tumor tissues and SCLC cell lines, MDK was expressed regardless of the subtype, and both non-NE- and NE-type SCLC cells were sensitive to MDK inhibition. These results suggest that MDK may be a versatile therapeutic target for SCLC. Nevertheless, further studies are required to clarify the association between MDK expression and SCLC characteristics.

Upregulated MDK expression in tumor tissues is associated with tumor growth in many cancers, including non-SCLC (NSCLC), hepatocellular carcinoma, and bladder cancer [17, 24, 25]. Furthermore, compared with healthy controls, patients with several cancers exhibit upregulated s-MDK expression, which is useful as a non-invasive biomarker for diagnosis and disease progression [17, 35–39]. Herein, we found increased s-MDK levels in patients with SCLC for the first time and observed a correlation between increased s-MDK levels and tumor burden in SCLC. Mostly, SCLC is diagnosed at an advanced stage, and owing to its high malignant potential, prompt treatment is often required. Thus, determining s-MDK levels can be helpful for the clinical diagnosis of SCLC. Furthermore, we found that s-MDK levels decreased with treatment in patients with high s-MDK secretion, and in one case (Fig. S1a), increased s-MDK levels were detected before disease progression based on imaging and tumor markers, indicating that it might be a predictive factor for disease progression during treatment.

MDK is involved in cancer cell proliferation, survival, migration, and resistance to chemotherapy via the PI3K/AKT and MAPK pathways [27, 40, 41]. Herein, MDK activated the AKT pathway in SCLC; however, its correlation to the MAPK pathway remained unclear. Hao et al. reported that MDK inhibition by iMDK suppressed only the PI3K/AKT pathway in NSCLC; however, the reason why the MAPK pathway was not inhibited remained unclear [25]. In our study, P-ERK was upregulated in H69 after MDK suppression, which might be compensated for by the downregulation of PI3K/AKT pathway. In MS1L cells, both the PI3K/AKT and MAPK pathways were enhanced after MDK overexpression, which indicated that pathway inhibition or activation might be cell-context-dependent.

We observed the combined antitumor effects of iMDK and CDDP in vitro and in vivo. Moreover, MDK and the PI3K/AKT pathway were upregulated in the CDDP-resistant SCLC cell lines. CDDP is one of the chemotherapeutic drugs currently used for SCLC; however, acquired resistance to this drug affects treatment efficacy. MDK is overexpressed in several cancers and mediates resistance to chemotherapeutic drugs via several mechanism [27]. MDK renders glioma cells resistant to tetrahydrocannabinol and inhibits autophagy-mediated cell death activation via the AKTmTORC1 pathway [42]. In gastric cancer, MDK overexpression upregulates P-AKT and P-ERK expression, which induces resistance to adriamycin [43]. Ying et al. reported that PI3K/AKT pathway activation was associated with resistance to chemotherapy in SCLC [32], indicating that PI3K/AKT pathway suppression might be the main reason why MDK inhibition in the present study enhanced the efficacy of CDDP.

Nevertheless, the present study had several limitations. First, owing to the small number of IHC evaluations and SCLC cell lines, concluding that no correlation exists between MDK expression and SCLC subtypes is difficult. Second, the age of control population for s-MDK measurement was relatively less than that of patients with SCLC included in this study. Reportedly, s-MDK gradually decreases with age in healthy children; hence, the difference in age may have had affected the results [44]. Third, owing to the small sample size and variable disease stages of patients whose MDK was measured, evaluating the correlation between s-MDK and prognosis was difficult. Lastly, although ICI combined with chemotherapy has become one of the standard therapies for SCLC, we did not evaluate the effect of MDK on the effects of ICIs. MDK suppressed CD8 T-cell functions by stimulating immunosuppressive myeloid-derived suppressor cells, which was driven in part by nuclear factor-κB in the TME, which resulted in resistance to ICI [22, 23]. If MDK inhibition improves T-cell functions and TME in SCLC, the combination of iMDK with ICI combined with platinum-based chemotherapy might be promising.

Altogether, this is the first study to evaluate the expression and functions of MDK in SCLC, and the finding that MDK inhibition enhances the efficacy of CDDP is crucial because it presents MDK as a potential therapeutic target for SCLC. The present findings show the pivotal role of MDK in SCLC, and thus, suggest MDK as a potential therapeutic target for SCLC treatment.

Acknowledgments

We thank M. Maeda (Department of Respiratory Medicine, Faculty of Medicine, Hokkaido University) for providing special support and assistance with this study.

Funding:

This study did not receive any specific grants from funding agencies in the public, commercial, or non-profit sectors.

Conflict of Interest:

The authors declare that they have no conflict of interest.

Ethics Statement:

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the institutional review board of Hokkaido University (identification number, 020-0379). All animal experiments were performed according to the protocols approved by the Institutional Animal Care Committee of Hokkaido University (approval number 23-0091).

Registry and the Registration No. of the study/trial: N/A

Consent to Participate:

Informed consent was obtained from all individual participants included in this study.

Consent to Publish:

All individual signed informed consent regarding publishing their data.

Data Availability Statement:

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.

Author Contributions:

Shotaro Ito and Jun Sakakibara-Konishi: Conceptualization, formal analysis, and drafting of the manuscript. All authors: Collecting the data and approving the results and conclusions.

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No competing interests reported.

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Midkine promotes tumor growth and attenuates the effect of cisplatin in small cell lung cancer

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Abstract

Purpose

Methods

Results

Conclusion

Figures

1. Introduction

2. Material and Methods

2.1 Reagents

2.2 Cell lines and establishment of CDDP-resistant lung cancer cell lines

2.3 ELISA for human MDK and tumor burden evaluation

2.4 Immunohistochemistry (IHC)

2.5 Antibodies and western blotting

2.6 Cell proliferation assay

2.7 Xenograft mouse model

2.8 Statistical analyses

3. Results

3.1 s-MDK reflects SCLC tumor burden

3.3 MDK regulates cell proliferation and migration in SCLC

3.4 MDK regulates intracellular signaling pathways in SCLC

3.5 MDK attenuated the antitumor effect of CDDP in SCLC

4. Discussion

Declarations

References

Additional Declarations

Supplementary Files

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