Metformin mediates AMPK/KIF1B signalling pathway to inhibit metastasis in bladder cancer cells

doi:10.21203/rs.3.rs-3466767/v1

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Metformin mediates AMPK/KIF1B signalling pathway to inhibit metastasis in bladder cancer cells

https://doi.org/10.21203/rs.3.rs-3466767/v1

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Background

To investigate the inhibitory effect of metformin on metastasis of bladder cancer cells and its potential mechanism.

Methods

The CCK-8 method and RTCAxCELLigence cell function analyzer were used to monitor and evaluate metformin activity changes and migration inhibition of SW780, RT4 and UMUC3. On this basis, Western blotting was used to evaluate the expression of AMPKα/P-AMPKα, mTOR, AKT/P-AKT and KIF1B antibodies in bladder cancer cells after adding metformin. In vivo, the metastatic inhibitory effect of metformin on bladder cancer was experimentally assessed by establishing a hematogenous lung metastasis model of bladder cancer in C57BL/6 mice by MB49 cells. Then the expression of AMPKα/P-AMPKα and KIF1B antibodies was again assessed in the tumour tissues of the two groups of mice using Western blotting.

Results

Low concentration of metformin can significantly inhibit the proliferation of SW780 and UMUC3, and a high concentration of metformin can significantly inhibit the proliferation of RT4. The IC50 of the three cells was 26.0 ± 1.4 mM, 32.9 ± 5.3 mM and 20.0 ± 3.4 mM, respectively. The migration of SW780 and UMUC3 was significantly inhibited by metformin when the concentration of metformin was more than 5MM and the time of action was more than 72h (P < 0.05). After adding metformin, P-AMPK was increased in RT4 and UMUC3, and the expression of KIF1B, AKT and mTOR antibodies was decreased. In vivo, The mean time of tumour formation in the metformin group was 34.5 ± 8.3 days, significantly longer than in the control group (24.8 ± 3.7 days, P = 0.035). In addition, the median survival time of mice in the metformin group was 40 days (P = 0.016). Compared with the control group, p-AMPK was up-regulated, and KIF1B was down-regulated in the metformin group.

Conclusions

Metformin can effectively inhibit the proliferation, migration, and invasion of SW780 and UMUC3 cells in vitro. Metformin can inhibit the migration of MB49 cells in vivo and increase mice's survival time. The mechanism of inhibiting the migration of UMUC3 in vitro and MB49 in vivo may be mediated by the AMPK pathway, which directly or indirectly inhibits the expression of its downstream KIF1B gene by activating P-AMPK.

Metformin

bladder cancer

phosphorylation

metastasis

AMPK signalling pathway

Bladder cancer(BC) is one of the ten most common cancers in the world[1]. Pathologically, bladder cancer can be divided into non-muscle-invasive BC and muscle-invasive BC. Clinically, approximately 70% of patients with BC are initially diagnosed with non-muscle invasive BC[2, 3]. Radical cystectomy with pelvic lymphadenectomy is the standard of care for muscle-invasive BC. However, up to 50% of patients are still at risk for postoperative recurrence and metastasis, with distant recurrence most likely in the lymph nodes, lungs, liver, and bone [2, 4]. Neoadjuvant cisplatin-based chemotherapy before surgery can prolong overall survival in patients undergoing radical cystectomy for muscle-invasive bladder cancer. Although most patients with bladder cancer are sensitive to Cisplatin's chemotherapy regimen, its efficacy is not long-lasting [5].

Clinical studies on patients with type 2 diabetes have proved that metformin (MET) can prevent the development of malignant tumours to a certain extent, and the incidence of cancer patients treated with MET is reduced[6]. In a 5-year follow-up of newly diagnosed patients with type 2 diabetes, cancer-related mortality was significantly lower in those taking MET to lower blood glucose levels than in those taking sulfonamide or insulin [7]. In addition, a prospective study found that MET prolonged the recurrence after transurethral resection of BC [8]. However, the primary effect of MET is to lower blood glucose, and the mechanism by which it benefits cancer patients is not yet clear.

MET can inhibit the growth and proliferation of several cancer cells, including bladder cancer 5637, RT4, and T24 cells [9-11]. The mechanism by which MET inhibits prostate cancer, breast cancer, and BC is that AMP-activated protein (AMPK) regulates ATP and NADPH levels, which in turn regulates cell growth [12]. MET can inhibit the proliferation of T24 by inducing apoptosis through mTOR signalling pathway [11, 13]. Furthermore, it can inhibit migration, and invasion and increase apoptosis of T24 and 5637 in in vitro experiments by down-regulating STAT3-related signalling pathway [14]. MET induces apoptosis or cell cycle arrest by activating multiple signaling pathways, exerts anti-bladder cancer effects and enhances the efficacy of targeted therapies and chemotherapeutic agents on MB49, T24, and UMUC3 [15, 16]. Previous reports in the literature on the mechanism of action of MET in inhibiting BC are mostly elaborated by inhibiting the proliferation of BC cells. The aim of this study was to verify whether there are more relevant mechanistic pathways for MET to inhibit BC cell migration and whether MET could inhibit the metastasis of BC cells in vivo, and to explain the potential molecular mechanisms of action.

Reagents and cell culture

SW780, RT4 and UMUC3 were provided by the Chinese Academy of Sciences cell bank in Shanghai, and Shanghai Yingwan Biotechnology Co., LTD, provided MB49. SW780, UMUC3, and RT4 were cultured using medium RMPI-1640(Corning, USA) containing 10% FBS(Corning, USA), 1% L-Glutamine(Corning, USA), and 1% penicillin-streptomycin-amphibian B solution (Shanghai Beyotime Biotechnology Co., Ltd., Shanghai, China). MB49 was cultured using DMEM/HIGH GLUCOSE (Corning, USA) medium containing 10% FBS, glutamine, and 1% penicillin-streptomycin-amphibian B solution. The cells were cultured in a humid atmosphere at 37°C and 5%CO₂. The present study used MET (Bristol-Myers Squibb, China) and one-glo Luciferase Assay System (Promega, Beijing).

Lentivirus IV8N-LUCI transfection

After digestion of MB49 cells, they were resuspended using DMEM/HIGH GLUCOSE complete medium without tertiary antibodies, and a suitable number of cells were seeded into 24-well plates and cultured overnight in a humid atmosphere of 37 °C, 5% CO₂. The next day, IV8N-LUCI lentivirus (luciferase-labeled lentivirus, which can make tumor cells possess stable luminescence ability after stable transfection, provided by Shanghai GenePharma Co.,Ltd, China) was added into the cells under the condition of MOI=10, the final concentration of Polybrene was 10 μg/ml to assist transfection. The MB49-LV8 cell line stably expressing luciferase gene was obtained by screening the cells with Puromycin at a concentration of 5 μg/mL. The expression of luciferase gene in MB49-LV8 cells was observed under inverted Fluorescence microscope, for following experiments.

Assay for cell growth

A Cell Counting Kit-8 (CCK-8) assay (DOJINDO, Japan) was used to determine cell proliferation. Logarithmic growth phase cells were digested with trypsin 0.25% solution (Corning, USA), and a single cell suspension was prepared. The cell density was adjusted to about 6×10⁴/mL, and 100μL cells of the suspension were inoculated in each well of a 96-well plate and cultured in an incubator for 24h. After the cells adhered to the wall, the medium was discarded and 2.5, 5, 10, 20, and 40mM MET were added and incubated for 48h, respectively. Four replicates were set for each concentration, and a blank control group was set with the same volume of medium. After the treatment, each well received 10μL of CCK-8 solution, the 96-well plate was gently knocked to mix, and then incubated for 2h. The absorbance value at 450 nm was measured using an enzyme plate analyzer (Bio techne, USA). The inhibitory rate of each cell at different drug concentrations was calculated: inhibitory rate = (control well-experimental well)/ (control well-blank well) , where the control well was the well with cells without drug and the experimental well was the well with cells with drug, the blank hole is no medium and no drug, IC50 is calculated by SPSS.

Assay for cell transwell

Real Time Cellular Analysis (RTCA) xCELLigence (ACEA Biosciences Inc, USA) was used to determine cell migration. Logarithmic growth phase cells were digested with 0.25% trypsin, and a single cell suspension was prepared. Using the Cell Invasion Migration Plate 16(CIM-Plate 16), 165μl RMPI-1640 was added in the lower chamber by Pipette (two wells were set for each cell to add serum-free medium, the rest were added to serum-containing medium and MET serum-containing medium with concentrations of 2.5, 5 and 10 mmol/L, respectively) , 30 mL of serum-free RMPI-1640 was added into the upper chamber, and the medium was distributed evenly around the clapper. It was then equilibrated for 1 h in an incubator at 37 ° C with concentration of 5%CO₂. The detection steps relevant to this experiment were set up, and the device equilibrated for 1 h was placed on the RTCA DP Analyzer to start the baseline detection. After the baseline CIM plates were removed, 100 mL of cell suspension was added to the upper chamber wells, and the CIM plates were transferred to the RTCA instrument after 30 min of resting at room temperature, and the CI values were set to be detected every 15 min. The cell index was continuously monitored during the period, and the observation time was longer than 96 hours after adding cells.

Mouse model of blood pulmonary metastasis

Male C57BL/6 mice (4-5 weeks old; weithing~20g) were supplied by Hunan SJA Laboratory Experimental Animal Co. , Ltd. China. Mice were fed with food and water ad libitum and were fed at specific pathogen‑free conditions at 24˚C, 60% humidity and alternating 12h light/dark cycles. Before the experiment, A total of 14 athymic nude mice were included in the control(n=7) and metformin(n=7) groups. Each mouse was injected via tail vein with 0.1 mL of mouse bladder cancer MB49-LV8 cell suspension at a concentration of 1 × 10⁷ cells/mL. Mice in the metformin group were injected with tumor cells via the tail vein and given MET(1.5mg/mL) (17) daily via drinking water, while mice in the control group drank normal water. After inoculation, the state of mice was observed every day, and the weight of mice was weighed twice a week. 3 weeks after tumor cell inoculation, each mouse was observed by intraperitoneal injection of Luciferase 250 mg/Kg using a bioluminescence/fluorescence/X-ray triad small animal imaging system (Ivis Lumina LT, USA). After the fluorescence was observed in the lungs of mice by in vivo imaging, the tumor was successfully formed in the model of lung metastasis. Survival time was observed after tumor formation was found. After the mice were anesthetized with isoflurane, the mice were killed by cervical dislocation. Half of the lung and liver tissues were fixed in 4% formaldehyde solution and the other half were stored at -80°C. The pathological sections were prepared and stained with hematoxylin and eosin. The morphological and histopathology changes of the cells were observed under microscope.

Western blotting

Logarithmic phase cells were and digested using trypsin 0.25% solution to prepare a single cell suspension, and the cell density was adjusted to 1 × 10⁶/mL. The cells were inoculated in a 6-well plate at 1mL/well and cultured in an incubator for 24h. After the cells had attached to the wall, we discarded the medium and the cells were divided into control group and metformin group. The metformin group was treated with 5mmol/L metformin for 72h, and the control group was treated with the same volume of medium containing 10%FBS. The cells were collected, cell lysis buffer containing protease and phosphatase inhibitors was added, and the cells were lysed on ice for 15 min. After centrifugation at 12000rpm for 15min at 4°C, the supernatant was retained, and its protein concentration was determined using the bicinchoninic acid (BCA) (Thermo Scientific, USA) method. Then, 40 μg of protein was subjected to SDS–polyacrylamide gel electrophoresis, and the separated proteins were transferred to 0.45μm or 0.22μm polyvinylidene fluoride (PVDF) membranes, and blocked using 5% skimmed milk at room temperature for 2h. Primary antibodies were added [the dilution ratio of AMPK, AKT, P-AKT, mTOR and P-AMPK was 1:700(ZEN-BIOSCIENCE，China), the dilution ratio of KIF1B(ABCAM, USA) was 1:2000, and the dilution ratio of GAPDH(ZEN-BIOSCIENCE，China) was 1:5000], and placed in a refrigerator at 4°C for overnight incubation. The membranes were washed with 1×Tris-buffered saline-Tween 20 (TBST) three times for 10 min each time and added with labeled secondary antibody at room temperature for 2h. After washing with 1 × TBST three times (10min each time), ECL (Thermo Scientific, USA) exposure luminescence solution was added at a 1:1 ratio onto the PVDF membrane for luminescence development. The immunoreactive protein bands were analyzed using Image-J 1.x software (NIH, Bethesda, MD, USA). The relative level of each protein was represented by the ratio of the gray level of target protein band to the gray level of internal reference, GAPDH.

Statistical analysis

Data was expressed as the mean +- SEM of three independent experiments performed in triplicate, with paired T-test applied before and after the use of MET. The rest were compared using an independent-sample t-test, with IBM SPSS Statistics 17.0 indicated that there was a statistical difference (P<0.05), and mouse survival time was measured using the Log-rank (Mantel-Cox) test. Data results were analyzed graphically using Adobe Photoshop CS 5(Adobe Photoshop, USA) and GraphPad Prism 5 (GraphPad inc., La Jolla, CA, USA).

Metformin inhibits the proliferation of SW780, RT4 and UMUC3 cells in vitro

At low concentration(5mM), MET could significantly inhibit the activity of SW780(P<0.001) and UMUC3(P=0.011), high concentration of MET (40mM) significantly inhibited the growth of RT4 (P<0.001) (Fig. 1A). The IC50 of MET on SW780, RT4 and UMUC3 cells were 26.0±1.4 mM, 32.9±5.3 mM and 20.0±3.4 mM, respectively. (Fig. 1B)

Metformin inhibits the migration of SW780 and UMUC3 cells in vitro

SW780 at different concentrations of MET showed a certain degree of inhibition from 24h on compared with the control group. The CI value of the medication group at the concentrations of 5 mM and 10 mM was significantly lower than that of the normal group, and decreased by about half After 48 h, the decrease was more obvious and showed a gradient, especially at the concentration of MET>5 mM and action time in 72h(P=0.014) (Fig. 2A). UMUC3 reacted with different concentrations of drugs in a concentration-dependent manner at the beginning of 24h, and the inhibitory effect of different drug concentration gradients became more obvious as time went on, when the MET concentration was > 5 mmol/L and 72 h, it was the most significant (P=0.011) (Fig. 2B). RT4 cells did not transfer, and the effect was not obvious after adding MET (Fig. 2C).

The AMPK/KIF1B pathway is inhibited by metformin

Detection of protein expression in SW780, RT4, and UMUC3 by Western blot (Fig. 3A). After adding MET, P-AMPK was activated to some extent in RT4(P=0.196) and UMCU3(P<0.001). In addition, the expression of KIF1B was inhibited in RT4(P=0.006) and UMUC3(P=0.008) cells, but not in SW780 cells (Fig. 3B-3C).

In RT4 (P=0.020/ P=0.100) and UMUc3(P=0.371/ P=0.028) cells, the AKT/P-AKT signaling pathway was inhibited after the addition of MET. Moreover, which downregulates the mTOR signaling pathway in RT4(P=0.002) and UMUC3(P=0.050).

Therefore, we hypothesized that MET inhibits UMUC3 metastasis by activating P-AMPK and inhibiting its downstream mTOR signalling pathway, thereby suppressing KIF1B expression (Fig. 3D).

Metformin inhibits the hematogenous metastasis of bladder cancer to the lung in a mouse model of lung metastasis

Compared with the control group, which had a tumorigenesis time of 24.8±3.7 days, the average tumorigenesis time of mice in the metformin group was 34.5±8.3 days (Fig. 4A-B), which was slower (P=0.035). By dissecting the mice, we found that the lungs of control mice were filled with nodules 1-3cm in size and had lost their normal lung morphology (Fig. 4C). The median survival time was 40 days in the metformin drinking group compared with 28 days in the control group (Fig. 4D), showing a significant prolonging effect(P=0.016).

Further study, in vivo, MET also increased the expression of P-AMPK (P= 0.006) and inhibited the expression of KIF1B(P= 0.023)(Fig. 4E) .

In this study, we found that MET not only inhibited the proliferation of SW780, RT4 and UMUC3, but also inhibited the migration of SW780 and UMUC3. In addition, the degree of inhibition increased with the time and concentration in SW780 and UMUC, when the MET concentration >5 mmol/L, the inhibition of metastasis was more obvious(P < 0.05). The pathway to inhibit metastasis may be through activation of P-AMPK, which inhibits the expression of mTOR and thus the driver KIF1B. It is also possible that AKT/mTOR signaling pathway may be downregulated to inhibit protein synthesis and cell metastasis, which may play a role in inhibiting bladder cancer cell metastasis. In vivo, the tumor formation time(P = 0.035) and survival time (P = 0.016) were significantly prolonged after MET was added. In this study, P-AMPK was activated by the addition of MET, which inhibited KIF1B thereby inhibiting the migration and invasion of UMUC3, as well as the migration of MB49 cells in vivo. It is suggested that the inhibition of BC cell migration by MET may also revolve around the KIF1B signaling pathway, which is a possible mechanism related to metformin treatment of BC.

Mammalian Sirolimus target protein (mTOR), one of the therapeutic targets of BC, is also involved in the formation of eukaryotic biological signaling networks that coordinate cell growth. If abnormal activity of mTOR signaling pathway can lead to uncontrolled cell proliferation, it can induce various cancers, such as breast cancer, lung cancer, prostate cancer, bladder cancer, etc. [18, 19]. In the AMPK/mTOR signaling pathway, AMPK is activated when ATP levels drop or the cell's energy needs increase, thereby downregulating the mTOR gene, inhibiting protein synthesis and cell growth through this signaling pathway, induce autophagy and maintain homeostasis [19]. In recent studies, tetrandrine and 10-hydroxycamptothecin have been shown to induce apoptosis and autophagy in bladder cancer cells by down-regulating ATP levels and thus activating the AMPK/mTOR signaling pathway[20, 21]. In this study, MET inhibited the metastasis of UMUC3 cells and activated P-AMPK (P = 0.011) and mTOR (P = 0.050) when metformin was added, indicating that MET inhibited the proliferation of RT4 and UMJUC3 cells, AMPK/mTOR signaling pathway may also inhibit the proliferation and metastasis of bladder cancer.

Here, we found that kinesin superfamily proteins (KIFs), a group of microtubule-dependent motors, play a functional role in cell division and intracellular transport of organelles, protein complex and vesicles. KIF1B, a member of KIFs, plays an important role in the apoptosis process and transformation and progression of malignant cells. It may be involved in the invasiveness of human cancers, such as gastric cancer, liver cancer and ovarian cancer [22-24], but its role in BC is unclear. The mechanism of action in renal cancer may be the involvement of KIF1B expression via AKT pathway [22]. In this experiment, we found that P-AMPK protein expression was activated after the addition of metformin to UMUC3, the expression of P-AKT was inhibited (P = 0.028). KIF4A, a member of the KIFs family, was found to be highly expressed in BC (P < 0.01). After knockdown of KIF4A, the proliferation and migration of T24 and 5637 cells were significantly decreased, which was related to Akt pathway, it is also associated with cellular EMT [25, 26]. KIF4A has been reported to be overexpressed in multiple tumors and also to be associated with patient prognosis in a variety of cancers, such as colon cancer, lung cancer, and hepatocellular carcinomas [27-29]. Another study found that knockout of KIF11, a member of the KIFS family, can inhibit the invasion and migration of breast cancer, a mechanism of action also involved in the EMT process and AMPK, AKT [30]. In this study, we found that the expression of KIF1B was significantly downregulated by MET in BC cells RT4 (P = 0.053) and UMUC3 (P = 0.045). On this basis, it also inhibited the migration and invasion of BC cells. In addition, P-AKT expression in UMUC3 was also significantly inhibited by MET (P = 0.028). In this study, AKT signaling pathways were also inhibited after inhibition of KIF1B expression by addition of MET, suggesting that the mechanism after inhibition of KIF1B in BC is also associated with AKT pathway.

In vivo study, compared with the control group, the addition of MET prolonged the tumor formation time (P = 0.035) and the survival time (P = 0.016). Moreover, the addition of MET activated P-AMPK (P = 0.006) and suppressed KIF1B (P = 0.023) in mouse lung xenografts, it is also possible that MET inhibits the hematogenous lung metastasis of BC in C57BL/6 mice through this pathway. KIF4A has been found to promote the secretion of the tumor chemokine CXCL5 to influence the recruitment of MDSCs to tumor sites to influence the immune microenvironment in the tumor tissues of BC [26]. It has been reported that MET are involved in the regulation of human MDSC-mediated immunosuppression in vitro and in vivo, mainly through AMPK activation followed by induction of MDSC activation. MET therapy has been shown to upregulate sausage homologue 1(DACH1) through P-AMPK, thereby reducing migration of MDSCS in the oesophageal squamous-cell carcinoma [31, 32]. Other studies have shown that MET can inhibit their activity in MDSCS by activating AMPKα and downregulating gene expression in the Hypoxia-inducible factors 1-α (HIF-1 α) pathway, this eventually leads to an overall decrease in MDSC inhibitory activity [33]. In addition, the role of MET in cancer may be mediated by activation of NK cells [34, 35], influence macrophage polarization (by increasing the expression of M1-related cytokines, attenuating the expression of M2-related cytokines and polarizing macrophages to an anti-tumor phenotype) [36, 37]. These results suggest that MET may also act by targeting the tumor's immune microenvironment.

This study found that MET could effectively inhibit the proliferation, migration, and invasion of SW780 and UMUC3 in vitro, and inhibit the migration and invasion of MB49 in vivo, and effectively prolong the survival time of mice. The mechanism of its inhibition of the migration and invasion of UMUC3, as well as the migration and invasion of MB49 in mice in vivo, may be through mediating the AMPK/KIF1B signaling pathway. However, more clinical, and basic experimental studies are needed to investigate the safe dose range of MET as an adjuvant drug in patients with BC and how to use it correctly in the clinic.

Ethics approval and consent to participate: All experimental protocols were approved by the Experimental Animal Ethics Committee of Guangxi Medical University. As well as all methods were carried out in accordance with Comply with the 《Guiding Opinions on the Kind Treatment of Laboratory Animals》issued by the Ministry of Science and Technology of the People's Republic of China and the national standard of the People's Republic of China, GB/T35892-2018, 《Guidelines for Ethical Review of the Welfare of Laboratory Animals》.

Consent for publication: Not applicable.

Availability of data and materials: Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.

Competing interests: The authors declare that they have no competing interests.

Funding: Supported by the "139" program training project for the cultivation of high-level and key talent in Guangxi medicine (G201903036), Open Project of Key Laboratory of Biomolecular Medicine Research in Guangxi Universities (GXBMR201605),Guangxi Colleges and Universities Key Laboratory of Biological Targeted Diagnosis and Treatment (GXSWBX201501), Guangxi Natural Science Foundation Project (2018GXNSFAA138061), Scientific Research and Technology Development Project, Wuming District, Nanning (20183203-3), Guangxi Medical & Health Appropriate Technology Development and Promotion Application Project (S2019052).

Authors’ contributions:

TBW, 1) Concept and design of the study; 2) Collection and assembly of data; 3) Data acquisition, analysis, and interpretation; 4) Manuscript writing; 5) Final approval of the manuscript

FH, 1) Data analysis and interpretation; 2) Manuscript writing; 3) Final approval of the manuscript.

MYZ，1) Data analysis and interpretation; 2) Manuscript writing; 3) Final approval of the manuscript.

YT, 1) Concept and design of the study; 2) Administrative support; 3) Data analysis and interpretation; 4) Manuscript writing; 5) Final approval of the manuscript.

QW, 1) Concept and design of the study; 2) Provide research data; 3) Data analysis and interpretation; 4) Manuscript writing; 5) Final approval of the manuscript.

All authors read and approved the final manuscript.

Acknowledgements: Not applicable

Author information

Corresponding Author:

Qi Wang, PhD, Key Laboratory of Early Prevention and Treatment for Regional High-Frequency Tumor, Ministry of Education, Nanning, China. [email protected]

Yong Tang, MD, PhD, Department of Urology, Wuming Hospital of Guangxi Medical University, Nanning, 530199, China. [email protected], Phone: +86 (771) 636-3896

Authors’ ORCID:

Yong Tang ORCID: 0000-0001-9214-4412

Wang Qi ORCID:0000-0002-9453-2547

Tian-bin Wen ORCID: 0009-0004-6468-4664

Fei Huang ORCID: 0009-0005-0850-884X

Ming-yong Zha ORCID: 0009-0006-6639-0293

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Metformin mediates AMPK/KIF1B signalling pathway to inhibit metastasis in bladder cancer cells

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