Exosomes from Nischarin-Expressing Cells Reduce Breast Cancer Cell

Published OnlineFirst January 11, 2019; DOI: 10.1158/0008-5472.CAN-18-0842

Cancer Molecular Cell Biology Research

Exosomes from Nischarin-Expressing Cells Reduce Breast Cancer Cell Motility and Tumor Growth Mazvita Maziveyi1, Shengli Dong1, Somesh Baranwal2, Ali Mehrnezhad3, Rajamani Rathinam4, Thomas M. Huckaba5, Donald E. Mercante6, Kidong Park3, and Suresh K. Alahari1

Abstract

Exosomes are small extracellular microvesicles that are secre- secreted by Nischarin-expressing tumors inhibited tumor ted by cells when intracellular multivesicular bodies fuse with growth. Expression of only one allele of Nischarin increased the plasma membrane. We have previously demonstrated that secretion of exosomes, and Rab14 activity modulated exosome Nischarin inhibits focal adhesion formation, cell migration, secretions and cell growth. Taken together, this study reveals a and invasion, leading to reduced activation of focal adhesion novel role for Nischarin in preventing cancer cell motility, kinase. In this study, we propose that the tumor suppressor which contributes to our understanding of exosome biology. Nischarin regulates the release of exosomes. When cocultured on exosomes from Nischarin-positive cells, breast cancer cells Signiﬁcance: Regulation of Nischarin-mediated exosome exhibited reduced survival, migration, adhesion, and spread- secretion by Rab14 seems to play an important role in con- ing. The same cocultures formed xenograft tumors of signiﬁ- trolling tumor growth and migration. cantly reduced volume following injection into mice. Exosomes See related commentary by McAndrews and Kalluri, p. 2099

Introduction rins to attach to extracellular matrix (ECM) proteins (6, 7). Each Nischarin, or imidazoline receptor antisera-selected (IRAS) integrin has designated ligand(s), and decreased expression of protein, is a protein involved in a number of biological processes. the ligand or receptor affects focal adhesion number. Integrins The Nisch gene is located on chromosome 3p21, which is fre- also bind to fibronectin-coated exosomes (8). Exosomes are quently lost in cancers (1). Most notably, Nischarin is an integrin smaller microvesicles (30–200 nm in diameter) secreted from a5b1 binding protein known to affect cell migration by antago- cells when multivesicular bodies (MVB) fuse with the plasma nizing the actions of cell signaling proteins that contribute to membrane (9–12). Although Nischarin's role has yet to be tumor cell migration and invasion (2). Furthermore, Nischarin linked to exosomes, previous studies have shown that the has also been shown to affect cytoskeletal reorganization, mainly Nischarin–Rab14 interaction promotes the maturation of þ by inhibiting Rac-induced lamellipodia formation (2). Consistent CD63 endosomes (13). Nischarin is an effector of the GTPase with this, Nischarin's inhibition of cell migration has been linked Ras-related protein Rab-14 (13). Although Rab14 is involved in to other proteins (3–5). vesicle sorting and trafficking (14), only one report has iden- During cell migration, cells adhere to its extracellular envi- tified Rab14 function in breast cancer exosomes (15). Nischarin ronment through focal adhesions. These complexes use integ- directly interacts with Rab14 to effect intracellular Salmonella survival (13). In the presence of Nischarin, there is triple

1 colocalization between the late endosome and exosome marker Department of Biochemistry and Microbial Sciences, Louisiana State University CD63, Rab14, and Nischarin (13). Health Sciences Center School of Medicine, New Orleans, Louisiana. 2Center of Biochemistry and Microbial Science, Central University of Punjab, Bathinda, While it is known that MVBs fuse with the plasma membrane Punjab, India. 3Division of Electrical and Computer Engineering, Louisiana State just before exosome release, the physiologic consequences of University, Baton Rouge, Louisiana. 4Wayne State University, Detroit, Michigan. this have yet to be determined in the breast cancer microen- 5Department of Biology, Xavier University of Louisiana, New Orleans, Louisiana. vironment. Furthermore, the proteins responsible for the MVB– 6 Louisiana State University Health Sciences Center School of Public Health, plasma membrane fusion are not well characterized. We Louisiana State University Health Sciences Center School of Medicine, New hypothesized that Nischarin may affect the migration of cancer Orleans, Louisiana. cells by controlling exosome release. Exosomes from 231 cells Note: Supplementary data for this article are available at Cancer Research promoted migration of 231 cells, while exosomes from 231 Online (http://cancerres.aacrjournals.org/). Nisch cells inhibited migration. These effects were due to the Corresponding Author: Suresh K. Alahari, LSUHSC School of Medicine, 1901 decreased number of exosomes released by 231 Nisch cells. In Perdido Street, New Orleans, LA 70112. Phone: 504-568-4734; Fax: 504-568- contrast, active Rab14 promotes exosome secretion and cell 2093; E-mail: [email protected] growth.Insummary,ourstudyhighlights the antitumoral doi: 10.1158/0008-5472.CAN-18-0842 function of Nischarin expression mediated by exosome- 2019 American Association for Cancer Research. dependent secretions in breast cancer.

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Materials and Methods Animal studies All mouse experiments were performed in accordance with the Cell culture protocols approved by the Institutional Animal Care and Use MDA-MB-231, MDA-MB-231 Nischarin, MDA-MB-231 GFP, Committee of the Louisiana State University Health Sciences MDA-MB-231 Rab14, MDA-MB-231 Rab14 S25N, MDA-MB- Center (New Orleans, LA). Four- to 10-week-old female Prkdc 231 Rab14 Q70L, MCF7, BT20, T47D, MDA-MB-468, and scid mice were used in the xenograft studies (4 mice per group). SUM185, and MCF7 cells were cultured in DMEM at 37 C, The exosomes used for all mouse coinjections were isolated 5% CO supplemented with 10% FBS and 1% penicillin/ 2 from 1.8 g of mouse mammary tumor. Therefore, the approxi- streptomycin (Gibco). MCF10A cells were cultured in þ þ mate number of Nisch / exosomes isolated were 1 108 while DMEM/F12 supplemented with 5% horse serum EGF, hydro- þ the number of Nisch / exosomes was 1.9 108. After exosome cortizone, choleratoxin, insulin, and penicillin/streptomycin. isolation, animals were either injected with exosomes only, MDA-MB-231 Nisch, MDA-MB-231 GFP, MDA-MB-231 þ þ þ þ þ 1 106 viable Nisch / or Nisch / cells alone, or Nisch / Rab14, MDA-MB-231 Rab14 S25N, and MDA-MB-231 Rab14 þ þ þ or Nisch / cells that were cocultured with the Nisch / or Q70L were prepared as described previously (4). Briefly, þ Nisch / exosomes for 4 days. Tumor growth was assessed every 231 Nisch cells were generated by amplifying human NISCH. three days with calipers and tumor volume was calculated as The 4545 base pair PCR product was then cloned into a p length width2/6. pCDH-CMV-MCS-EF1-copGFP vector. The viral particles were generated in HEK-293T cells along with the pCDH- GFP-Nisch plasmid. The supernatants containing the lentiviral Isolation of exosomes particles were collected 48 hours later, concentrated, then A total of 3 106 cells were seeded in T175 flasks and cultured reconstituted in serum-free media. The MDA-MB-231 GFP for 48 hours. The medium was centrifuged at 300 g for cells were generated similarly, except there was no cloning of 10 minutes, and then the supernatant was collected and centri- NISCH into the pCDH-CMV-MCS-EF1-copGFP vector. The fuged again at 2,000 g for 20 minutes. This was repeated again cells were sorted by FACS for GFP selection. Low passage for two more centrifugations both at 10,000 g for 30 minutes cells were used for all the experiments and the cell line purity and 1 100,000 g for 70 minutes. The supernatant was then was verified every two months using appropriate markers of discarded and the pellet was centrifuged with PBS for 70 the cell type. Transfected cells were selected using the antibi- minutes at 1 100,000 g. The pellet was finally resuspended otic puromcyin. in PBS for subsequent studies. To isolate exosomes from þ þ þ þ þ Nischarin WT ( / ), Nischarin HET ( / ), and Nischarin Null whole tumors, we harvested breast tumors from Nisch / and þ ( / ) animals were generated as described previously (16). Nisch / mice as described previously (17). Tumors were Briefly, exons 7 to 10 of Nischarin were deleted, and the disintegrated and incubated with collagenase for 3 hours. The resulting animals were intercrossed with animals expressing samples werecentrifuged for 5 minutes at 200 g.Thesuper- the mouse mammary tumor virus-polyma middle T transgene. natant was then used for the exosome isolation procedure by For mouse genotyping, mouse tail genomic DNA was extracted ultracentrifugation. and amplified by PCR and electrophoresed on 2% agarose gels. The Beckman Couter DelsaNanoC instrument was used to þ þ þ Primary WT-PyMT (Nisch / ), HET-PyMT (Nisch / ), and measure diameter and molecular weight. The samples were dilut- / Null-PyMT (Nisch ) cells were isolated as described previ- ed in ddH2O, added to a cuvette, and inserted into the device. ously (17). Briefly, the mammary tumors were isolated and cut This instrument uses Photon Correlation Spectroscopy (PCS) to into small pieces with a razor blade and scissors. The tissues analyze the molecular weight of the sample as it relates to the were incubated with collagenase for 2 hours to allow for a and b ion particles in the solution. Basically, when laser light is enzymatic dissociation of the tissue. The resulting material was directed toward the particle, light will scatter in different direc- ultracentrifuged to remove debris and blood. The following tions. The intensities observed by the machine are a result of conditions were used for cell culture experiments of cells that the movement of ion particles due to Brownian motion. For were seeded on ECM proteins, 10 mg/mL of fibronectin (BD particle counting, the Malvern NanoSight, a Nanoparticle Biosciences) was prepared in PBS. Bovine collagen 1 (BD Tracking Analysis instrument was used. The samples were diluted 2 Biosciences) was added to each well at 0.16 mL/cm .TheECM in ddH2O and added to the flow system with a syringe. As the proteinswereaddedtothewellsandplacedonarockerfor samples pass through the flow system, the exosomes are visual- 2 hours at room temperature then washed two times with warm ized, counted, and characterized. PBS. The cells were seeded onto the wells immediately after washing with PBS. Labeling of exosomes þ þ þ Exosomes were isolated from Nisch / and Nisch / Cell line authentication tumors and resuspended in PBS. For CD63 labeling, the MDA-MB-231 cells were obtained from ATCC and Nischarin CD63 antibody was added at a concentration of 1:100 and expression in these cells was maintained by puromycin selection. incubated for 30 minutes at 37C as described previously Nischarin expression was monitored by immunoblotting using (18). The exosomes were rinsed in 0.5% BSA-PBS and cen- anti-Nischarin antibody. Cells were not used beyond passage five trifuged at 100,000 g for 1 hour at 4C. The secondary and Mycoplasma was tested for all cell lines at least once every antibody (Alexa Fluor 594) was added at a 1:200 concentra- 6 months. The primary cells prepared from PyMT tumors were tion for 30 minutes at 37C. The exosomes were washed again tested every time for Nischarin truncation genotype, PyMT expres- by centrifugation, and the pellet was resuspended with PBS. sion by genomic PCR approach. The primary cells were never used Exosome images were captured using the Leica DMi8 confocal beyond passage three. microscope.

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Antibodies For phalloidin staining, cells were washed two times with PBS, Antibodies and dilutions were used as follows: mouse and fixed in 3.7% formaldehyde for five minutes. The cells were monoclonal anti-vinculin (Sigma; 1:5,000), mouse monoclonal washed three times with PBS for five minutes each and were anti-paxillin (BD Biosciences; 1:5,000), rabbit monoclonal anti- stained with 50mg/mL of a fluorescently conjugated phalloidin flotillin (Cell Signaling Technology; 1:100–500), mouse mono- (Sigma) for 30 minutes. After staining, the cells were washed three clonal anti-CD63 (Santa Cruz Biotechnology; 1:500), mouse times in PBS for 10 minutes each. The coverslips were mounted monoclonal anti-Rab14 (Santa Cruz Biotechnology; 1:100– with DAPI Fluoromount-G (Southern Biotech). 1,000), mouse monoclonal anti-Nischarin (BD Biosciences; CellProfiler was used to determine the number of focal adhe- 1:1,000), and phalloidin (Sigma; 1:100). sions per cell and the percentage of area covered by focal adhesions. After incubating the cells overnight on fibronectin, the cells were Cell proliferation assay fixed and stained with vinculin. Images of vinculin-stained cells For MTT cell viability assays, cells were seeded onto 96- were uploaded into CellProfiler and the Enhance or Suppress well plates for 24–48 hours with the various coatings. MTT (3- Features and Indentify Primary Objects modules were used to (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assess the focal adhesions. For the Identify Primary Objects mod- reagent (5mg/mL in PBS) was added to each well. After a 3.5- ule, the typical diameter used was 10–50. The threshold strategy hour incubation, the media/MTT was aspirated and a HCl/iso- was Global, the thresholding method was Robust Background. The propanol solution (0.1% NP-40) was added to each well. The lower bounds on threshold was then changed to 0.001. Images plate was incubated for 20 minutes and then the absorbance was were captured at 60 using the Nikon Eclipse Ti-S. measured at 565 nm. The results were normalized to medium alone control. Real-time PCR For cell counting experiments, 5,000 231 GFP, 231 GFP Nisch, Total RNA was isolated from cultured cells with TRIzol reagent þ þ þ Nisch / , and Nisch / cells were seeded onto 96-well dishes (Invitrogen). cDNA was generated from 2 mg of RNA using the and incubated for 24 hours. After 24 hours, representative images Invitrogen/Applied Biosystems/ABl High Capacity cDNA Reverse were captured at 10 with the EVOS XL Cell Imaging System. The Transcriptase Kit. For gel detection, samples were run on a 3% number of cells per milliliter were calculated using the Bio-Rad agarose gel. TC20 Automated Cell Counter. Stiffness with indentation Live-cell migration A cylindrical sample with 6-mm diameter was cut out from the For live-cell imaging, the cells were seeded onto 12-well tumor tissue and constrained by a PDMS mold. The sample was mounted inside a petri dish on top of an electronic scale (SPX622, plates and cultured at 37 Cwith5%CO2 overnight. The next day, a small wound was created on the corner of the well with a Ohaus), which is located on a motorized stage (H101E1F, PRIOR fi 10 mL pipette tip to stimulate migration, and live cell migration Scienti c). The motorized stage brings the sample up and pushes it fi was captured every hour for 19 hours. Max distance and mean through a xed circular indenter with 1-mm diameter, which velocity were calculated using the ImageJ plugins MTrackJ and produces a known surface displacement of the sample. The Chemotaxis Tool. Migration tracks were created by using the resulting force is measured simultaneously using the scale and X and Y coordinates from the MTrackJ plugin. These coordi- the force versus strain curve is extracted. To calculate the stiffness, nates were tracked by the microscope and registered at subpixel an indentation model is used considering the geometry of the m precision as the inverse of the zooming factor. For data repre- indenter (19). The stage is lifted with step size of 10 muntila sentation, the subsequent X and Y points were subtracted from force of 5 mN is applied on the sample. To avoid the adhesion the previous X and Y points. The represented axis boundaries effect (20), the average slope over the region between 50% and fi were selected on the basis of the cell that traveled the most 100% of the maximum indentation force is de ned as contact fi distance and standardized for each cell type. For example, the stiffness, k. The effective elastic modulus, Eeff can be de ned using 231 cell that traveled the furthest distance on the –x axis the standard relation (21) as shown in Eq. A. – reached (-313.333,-97.333), therefore the x axis for all 231 ( pffiffiffi p cells was set to -350 mm. Images were captured at 10 using the E ¼ pffiffiffik k eff 2 A ) Eeff ¼ ðAÞ Olympus IX81 and Nikon Eclipse Ti-S. ¼ pd2 d A 4 fl Immuno uorescence where, A is the projected contact area underneath the indenter and d fi Cells were washed two times with PBS and xed in 3.7% is the diameter of the circular indenter. formaldehyde for 10 minutes. Then, the cells were washed three Generally, the effective elastic modulus is a function of elastic times with PBS for 5 minutes each. The cells were then permea- modulus of both indenter and sample. However, because the bilized with 0.5% Triton-X100 for five minutes. The cells were elasticity of used indenter, Ei, is much larger than the elasticity of washed again three times with PBS then blocked for 1 hour with the sample, Es, this relation can be simplified as shown in Eq. B 2% BSA in PBS. After blocking, the coverslips were incubated upside down on the primary antibody overnight (vinculin or () n2 1n2 1 ¼ 1 s þ i CD63) at 4 C. After primary antibody staining, the cells were E E E ) ¼ð n2Þ ð Þ eff s i Es 1 s Eeff B washed three times for ten minutes each. The cells were then Ei >> Es incubated with the secondary antibody for 1 hour. Prior to mounting, the cells were washed twice with PBS for 10 minutes, where, ni and ns are Poisson's ratio of the indenter and the sample. then once with water. The coverslips were mounted with Fluor- Assuming that the sample is incompressible (ns ¼ 0.5), the elastic omount-G (Southern Biotech). modulus of the sample is extracted from contact stiffness as

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Nischarin Regulates Exosome Production

shown in Eq. C. the number of focal adhesions and the area covered by focal adhesions using CellProfiler. 231 Nisch cells had a reduction 3k in focal adhesion number and in the area covered by focal E ¼ ðCÞ s 4d adhesions (Fig. 1D). This suggests that the decrease in cell area is due to destabilized cell-matrix attachment. Several intracellular signaling pathways have been elucidated, but whether Statistical analysis Nischarin regulates attachment to specific components of the All experiments were repeated at least three times. Statistical ECM remains elusive. analyses involving only one factor were performed using Graph- Pad Prism 5.0 software using either a two-sample t test or one-way Nischarin alters exosomes' properties ANOVA. Experiments involving two factors were analyzed in SAS The ECM is a diverse mesh network of proteins that support cell (Version 9.4) with two-factor ANOVA with interaction. Pairwise attachment. The ECM stimulates PI3K activity, which results in mean comparisons were performed using the PDIFF option on the cancer phenotypes, such as anchorage-independent growth on LSMEANS statement of PROC GLM in SAS. The error bars indicate soft agar and protection from apoptosis (24, 25). Previous reports the SD from the mean (, P < 0.05; , P < 0.01; , P < 0.001; and show that culturing noncancerous cells in conditioned media , P < 0.0001). All experiments were repeated at least three from cancer cells promotes proliferation and migration of non- times. cancerous cells (26, 27). We previously determined that incubating noncancerous MCF10A cells with media from 231 cells promotes cell migration (28). Incubating the cells with media Results from 231 Nisch cells promotes a moderate amount of cell migra- Nischarin regulates cell attachment tion, but significantly less than media from 231 cells (28). These Detachment of cells from a matrix leads to anoikis, cell death results suggest that Nischarin is regulating the cancer cell secre- due to the loss of adhesion (22, 23). Because Nischarin reduces the tome. The possible factors involved in this regulation could be activation of proteins, such as FAK and Rac that contribute to cell matrix metalloproteases, cytokines, or secreted exosomes, to adhesion, we examined whether there was a decrease in cell name a few. Thus, we first examined whether Nischarin is regu- attachment in Nischarin-positive breast cancer cells seeded on lating the release of exosomes. different matrices. To perform this, we used our previously pub- Although each cell type secretes unique exosomes, CD63 and lished MDA-MB-231 cells stably expressing Nischarin (hereafter, flotillin1 are commonly used exosomal markers for mammary þ þ þ 231 Nisch) and Nisch / -Polyoma Middle T (PyMT), Nisch / - cells. Flotillin proteins function in a number of contexts, such as Polyoma Middle T (PyMT) and Nisch / - Polyoma Middle T endocytosis and cell signaling. We isolated exosomes from 231 (PyMT) cells (4). To determine whether Nischarin affects cell and 231 Nisch cells by differential ultracentrifugation. We vali- proliferation, we seeded MDA-MB-231 (hereafter, 231) and 231 dated flotillin1 expression in exosomes and cells by Western Nisch cells on 2D (directly on the tissue culture plate), Fibronec- blotting (Fig. 2A). Previous studies have shown that flotillin1 is tin, and collagen and measured cell proliferation using the MTT expressed in whole-cell lysates, as well as, in exosomes from 231 assay and automated cell counting. The human breast cancer cells cells (29). RNAs are also present in exosomes (30). We observed stably expressing Nischarin had a decrease in cell proliferation on the expression of HSP70, ITGA5, ITGA11, ITGAL, and ITGAV 2D, fibronectin, and collagen (Fig. 1A; Supplementary Fig S1A). mRNA in the exosomes (Fig. 2B). This further confirms that þ þ Similarly, we checked cell proliferation of Nisch / -PyMT, we have isolated exosomes. Very little amounts of Nisch mRNA þ Nisch / -PyMT and Nisch / -PyMT mouse tumor cells on 2D, and protein were found in the exosomes (Fig. 2B; Supplementary fibronectin, and collagen. Hereafter, these tumor cells are referr- Fig. S2A) and Rab27a is not detected in exosomes (Supplementary þ þ þ ed to as Nisch / , Nisch / , and Nisch / cells. On 2D, only Fig. S2B). Nisch / had a significant increase in proliferation. On fibronec- We examined the mean diameter of our exosomes (9–12). þ tin and collagen, Nisch / cells had significantly greater prolif- Photon correlation spectroscopy (PCS) indicated that our exo- þ þ eration compared with Nisch / (Fig. 1B; Supplementary Fig. somes are between 30 and 200 nm. Although not statistically S1B). Overall, Nisch / cells had the greatest amount of prolif- significant, exosomes from 231 cells had a mean diameter of 189 þ þ þ eration compared with Nisch / and Nisch / cells. In all of the nm while those from 231 Nisch cells have a lower mean diameter cell lines we tested, the presence of Nischarin in cancer cells led to of 172 nm (Fig. 2C). Because only small variations were observed a significant reduction in cell proliferation regardless of the matrix in vesicle size between 231 and 231 Nisch samples, we next (Fig. 1A and B). assessed changes in exosome quantity. Using PCS, we were able There are many possible contributors to reduced cell prolif- to analyze the molecular weight of the sample as it relates to a and eration. To determine whether the reduction in cell prolifera- b ion particles in the solution. Although not statistically signif- tion of Nischarin-positive breast cancer cells was due to cell icant, exosomes from 231 cells had a molecular weight of 1.49 attachment, we assessed cell spreading and focal adhesion 109 while those from 231 Nisch cells had a weight of 1.24 109 dynamics. 231 and 231 Nisch cells were seeded onto Fibro- (Fig. 2C). Because we did not see significant differences in the nectin, stained with phalloidin, and captured by immunoflu- diameter and molecular weight of 231 and 231 Nisch cells, we orescence microscopy. Cell area and aspect ratio were quanti- assessed particle number using the Nanoparticle Tracking Anal- fied with ImageJ. Nischarin decreased the average cell area and ysis instrument (Malvern NanoSight). 231 Nisch cells had a aspect ratio (Fig. 1C). This "shrinking" of cells is an indicator of decreased number of particles per frame (counted by the software) cancer cell death. To determine whether this reduction of cell and per milliliter (Fig. 2D). These results suggest that Nischarin- spreading is due to a decrease in focal adhesions, we stained positive cells produce less exosomes than Nischarin-reduced cells. cells with the focal adhesion marker vinculin, then determined Furthermore, we performed rescue experiments in Nisch

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2D Fibronectin Collagen A 0.8 0.4 0.5

0.4 0.6 0.3 ** 0.3 0.4 0.2 0.2 0.2 0.1 * 0.1 *** Absorbance at 562 nm Absorbance at 562 nm Absorbance at 562 nm 0.0 0.0 0.0

ch sch is N 31 Nisch 231 Ni -MB-231 2 - A - DA-MB-231 MDA-MB-231 MD M A-MB A-MB D D M M MDA-MB-231

Collagen 2D Fibronectin B *** 6 *** 6 8 * ** ** 6 *** 4 *** 4 4 2 2 2 Absorbance at 562 nm Absorbance at 562 nm Absorbance at 562 nm 0 0 0 T T M MT M T MT MT y + - P /- Py + - Py /- Py / -Py +/ - - - - -PyMT +/ - - - + + -PyMT /- -PyMT -/ -PyM + / h +/ + h h h h ch c sc c c isch is is i sch is isc N N isch i N N N N N N Nis

MDA-MB-231 CDMDA-MB-231 MDA-MB-231 Nisch MDA-MB-231 Nisch

Average area 4 800 4 150,000 3 600 3 * * 100,000 2 400 2 ** Area ** #FA per Area 50,000 Aspect ratio 1 200 1 % Area covered by FA 0 0 0 0 1 h 1 h c c -23 isch -23 s isch B N B-231 N MB 1 Ni M -MB-231 - - -231 Nis A DA-M A -231 -231 M D DA B -MB M B M MD A-M A-M MDA D DA-MB-23 D M M M

Figure 1. Nischarin decreases the attachment of breast cancer cells. A, The 231 (n ¼ 6) and 231 Nisch (n ¼ 6) cells were seeded onto a standard tissue culture surface (2D), fibronectin, or collagen for 48 hours. Cell proliferation was determined by the MTT assay. B, Nischþ/þ,Nischþ/,andNisch/ cells were seeded onto a tissue culture surface (2D), fibronectin, or collagen for 48 hours. Cell proliferation was determined by the MTT assay. C, The 231 (n ¼ 18) and 231 Nisch (n ¼ 21) cells were seeded on 10 mg/mL fibronectin for 24 hours and stained with phalloidin. The average area and aspect ratio were calculated using ImageJ (inset with a zoom is shown). Aspect ratio ¼ major axis/minor axis. D, Vinculin immunofluorescence staining of 231 and 231 Nisch cells (inset with a zoom is shown). Number of vinculin- positive focal adhesions (FA) per area and percentage of area covered by focal adhesion was calculated using CellProfiler. , P < 0.05; , P < 0.01; , P < 0.001.

knockdown cells to see whether we can reduce particle counts. To exosomes had an average diameter of 67nm, which was sig- þ do this, we introduced Nisch into previously published MCF7 nificantly lower than the Nisch / exosome size (98 nm; cells (4), which have a stable knockdown of Nisch. Introducing Fig. 2G). Furthermore, we found that for the same sized tumor, þ Nisch to knockdown cells reduced exosome numbers to control Nisch / exosomes had an average weight of 3.99 108,while þ þ levels (Supplementary Fig. S2C and S2D). These data indicate that Nisch / exosome samples had a significantly lower weight þ Nischarin-positive 231 cells secrete significantly fewer exosomes (1.86 108;Fig.2G).Finally,Nisch / and Nisch / exosomes while Nischarin-positive MCF7 cells secrete slightly fewer exo- have a slightly greater number of particles per frame, as well somes when compared with their Nisch-negative counterparts. as number of particles per ml, although not statistically signif- We also examined exosome number using whole mouse icant (Fig. 2H). Taken together, our results suggest that the þ þ þ tumor tissues. Exosomes secreted from Nisch / and Nisch / presence of Nischarin in cells promotes secretion of fewer and tumors are also CD63 positive (Fig. 2E and F). We assessed the smaller exosomes compared with cells that have nonfunctional þ þ diameter and molecular weight of exosomes. Nisch / tumor Nischarin.

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A B C

250 2.0

200 1.5 150 231 Exo 231 Cells 231 Nisch Cells 231 Nisch Exo 1.0 /1 E 09 E /1 231 Nisch Cells 100 w 231 Cells 231 Exo 231 Nisch Exo HSP70 M

Diameter (nm) 0.5 Flotillin ITGA5 50

ITGAL 0 0.0 h h ITGA11 231 sc Ni Nisc MB- -MB-231 1 Vinculin ITGAV A- MD MDA B-23 -MB-231 M Actin A D Nischarin M MDA- D

+/+ +/- 200 4 E Nisch -PyMT Nisch -PyMT Secondary only

150 3

100 2 per frame

50 *** (e+009)per mL 1 *** Number of particles Number of particles

0 0 1 ch 231 s -23 isch B N Ni MB- M A- A- D B-231 M MD M

DA-MB-231 DA- M M

FG H

150 0.5

Tumor Exo Tumor 4

Tumor Exo 300 Tumor ***

+/- 0.4 +/+ +/+ +/- *** 3 100 0.3 200 2 Nisch Nisch Nisch Nisch /1 E 09 E /1

w 0.2 50 (e+009) M per frame 100

Diameter (nm) Diameter 1 CD63 0.1 Number of particles Number of cells per mL 0 0.0 0 0 T T T T T T T T M MT M M M MT yM y y y yM y + P - PyM PyM P - Py P - / - -/- - /+ - -P - -P /+ - -P + -Py - +/ + + +/ -/ + +/- +/ +/- h h h h h c ch sch sc Vinculin ch ch s i isc isch isc isc is is is i N N Ni N N N N N N N

Figure 2. Characterization of exosomes from breast cancer cells. A, Western blot detection of ﬂotillin and vinculin (control) in 231 exosomes, 231 cells, 231 Nisch exosomes, and 231 Nisch cells. B, RT-PCR of NISCHARIN, ACTIN, HSP70, ITGA5, ITGA11, ITGAL,andITGAV mRNA in 231 and 231 Nisch exosomes, as well as 231 and 231 Nisch cells. C, Diameter and molecular weight analysis of exosomes from 231 (n ¼ 9) and 231 Nisch (n ¼ 9) cells with the DelsaNanoC. D, Number of particles per frame and per mL of 231 (n ¼ 6) and 231 Nisch exosomes (n ¼ 15) with the Nanosight Nanoparticle Tracking Analysis. E, Nischþ/þ and Nischþ/ exosomes were labeled with CD63 (inset with a zoom is shown). Scale bars, 10 mm. F, Western blot detection of CD63 and vinculin (control) in Nischþ/þ tumor exosomes, Nischþ/þ þ/ þ/ þ/þ þ/ tumors, Nisch tumor exosomes, and Nisch tumor cells. G, Diameter and molecular weight (Mw) analysis of exosomes from Nisch (n ¼ 3) and Nisch (n ¼ 3) tumors with the DelsaNanoC. H, Number of particles per frame and per mililiter of Nischþ/þ (n ¼ 6), Nischþ/ exosomes (n ¼ 11), and Nisch/ exosomes (n ¼ 3) with the Nanosight Nanoparticle Tracking Analysis. , P < 0.001.

Exosomes from Nischarin-positive cells reduce breast cancer substrates (Fig. 3A). The 231 Nisch cells have a much lower cell motility migratory capacity and do not reach 150 and 150 mmonthe Previous reports show that exosomes are fibronectin coated axes (Fig. 3B). There were more 231 Nisch cells that reached 50 and cancer cells migrate on them (8, 31). Thus, we investigated and 50 on the x-andy-axes when the cells were placed on whether the altered dynamics of exosomes from Nischarin- Fibronectin as well. Interestingly, migration on exosomes from positive cells affect cell motility. We have previously shown that 231 cells is higher than the migration on exosomes from 231 the presence of Nischarin in 231 cells inhibits cell migration on Nisch cells. The live cell tracking was further analyzed on gelatin and on matrices from NIH3T3 fibroblasts (28). MDA- ImageJ to detect the maximum distance and mean velocity. MB-231 or 231 Nisch cells were seeded on a dish coated with Maximum distance and mean velocity were significantly no coating (NC), fibronectin (as a positive control), exosomes reduced in 231 Nisch cells regardless of the coating but more from 231 cells, and exosomes from 231 Nisch cells and migra- pronounced on 231 exosomes. Interestingly, 231 cell migration tion was tracked every hour for 19 hours (Fig. 3A and B). Each on 231 Nisch exosomes is significantly lower than the on 231 individual line corresponds to one tracked cell. The 231 cells exosomes (Fig. 3C). Altogether, the cells migrated at a greater had more cells venture past 150 and -150 mmonthex-andy- distance and velocity on fibronectin, followed by on 231 axes when seeded on fibronectin compared with the other exosomes, then on 231 Nisch exosomes.

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A 231 Cells on NC 231 Cells on 231 Cells on 231 231 Cells on 231 250 Fibronectin Exosomes Nisch Exosomes C 250 250 Max distance 150 400 NC Fibronectin 300 -350 150 -50 -350 150 -350 150 -350 150 231 Exosomes 200 ** 231 Nisch Exosomes -250 -250 -250 -250 m 100 B 231 Nisch Cells on 231 Nisch Cells on 231 Nisch Cells on 231 Nisch Cells on 0

NC Fibronectin 231 Exosomes 231 Nisch lls ells h C 31 Ce c 250 250 250 Exosomes 2 is 250 231 N Mean velocity 0.3 NC -350 150 -350 150 -350 150 -350 150 Fibronectin 0.2 231 Exosomes -250 -250 -250 -250 ** 231 Nisch Exosomes

+/+ m m/min 0.1 Nisch+/+ Nisch Nisch+/+ Nisch+/+ Cells on NC Cells on Fibronectin Cells on Nisch+/+ Cells on Nisch+/- D 0.0 250 Exosomes Exosomes 250 ells C 250 250 231 Cells isch

231 N -350 150 -350 150 -350 150 -350 150 F Mean velocity -250 -250 -250 -250 0.20 *** NC +/- +/- +/- Fibronectin +/- Nisch Cells on Nisch Cells on Nisch Cells on 0.15 E Nisch Cells on NC ** +/+ Fibronectin Nisch+/+ Nisch+/- Exosomes Nisch Exosomes 250 0.10 Nisch+/- Exosomes 250 Exosomes 250 m m/min 250 0.05

0.00 -350 150 -350 150 -350 150 s /+ Cells Cell -350 150 + +/- h h c isc is -250 -250 -250 -250 N N

Figure 3. Exosomes from Nischarin cells reduce cell motility. The 231 (A) and 231 Nisch cells (B) were seeded onto NC, fibronectin, 231 exosomes, or 231 Nisch exosomes and live imaging was captured every hour for 19 hours (n ¼ 27) using an Olympus IX81 light microscope. Migration tracks were created using the X and Y coordinates from the MTrackJ plugin. C, Max distance and mean velocity were calculated using the ImageJ plugins MTrackJ and Chemotaxis Tool. D, Migration tracks of Nischþ/þ cells were seeded on NC (n ¼ 4), fibronectin (n ¼ 7), Nischþ/þ exosomes (n ¼ 11), or Nischþ/ exosomes (n ¼ 9). E, Migration tracks of Nischþ/ cells were seeded on NC (n ¼ 14), fibronectin (n ¼ 19), Nischþ/þ exosomes (n ¼ 27), or Nischþ/ exosomes (n ¼ 10). F, Mean velocity of Nischþ/þ and Nischþ/ cells on NC, fibronectin, Nischþ/þ exosomes, and Nischþ/ exosomes. , P < 0.01; , P < 0.001.

Next, we examined migration using mouse tumor exosomes Nischarin may lead the cells to increase release of exosomes for from animals with different Nischarin profiles. We investigated an added migratory advantage. whether the altered exosome characteristics from Nischarin-positive cells affect cell motility in mouse tumors. Fewer Nisch / Exosomes from Nischarin-positive cells reduce cell adhesion animals are viable; therefore, to maintain significant replicates for From the above data, we have established a potential role for þ þ þ our experiments, we only used Nisch / and Nisch / tumor exosomes in cell migration. Cells seeded on fibronectin have þ þ þ cells. Nisch / or Nisch / tumor cells were seeded on tissue greater dynamic adhesions and cell spreading. The presence of culture dishes coated with no substrate, fibronectin, exosomes dynamic adhesions determines that whether focal adhesion þ þ þ from Nisch / tumors, or exosomes from Nisch / tumors and machinery is able to disassemble and assemble in a highly motile migration was tracked every hour for 19 hours. The migration was cell. To understand how these exosomes reduce cell migration, we þ þ þ þ most dramatic for Nisch / and Nisch / cells on Nisch / examined cell adhesion to the exosomes and changes in focal exosomes (Fig. 3D and E). When mean velocity was assessed, adhesion numbers. MDA-MB-231 and 231 Nisch cells were þ there was a slight increase in the migration of Nisch / cells on seeded onto NC, fibronectin, 231 exosomes and 231 Nisch fibronectin, although not statistically significant. Furthermore, exosomes, and visualized the focal adhesion complexes. In addi- þ þ þ Nisch / cell migration on Nisch / exosomes is significantly tion, we analyzed the number of focal adhesions per cell using þ þ þ higher than on Nisch / exosomes (Fig. 3F). Nisch / exosomes CellProfiler. MDA-MB-231 cells displayed an increased number promoted greater cell velocity when compared with fibronectin of focal adhesions on all matrices, while the focal adhesions on þ þ and Nisch / exosome coating. The most significant changes 231 exosomes showed further increase in number. However, focal þ þ þ between Nisch / and Nisch / cells were seen with the exosome adhesions on 231 Nisch cells are significantly lower in number coatings (3F). Generally, exosomes from Nischarin-reduced cells (Fig. 4A–C). Furthermore, 231 cells seeded on 231 Nisch exo- increased cell migration compared with exosomes from somes showed less number of focal adhesions. As stated before, Nischarin-positive cells. The data suggest that the absence of Nischarin is an Integrin a5b1 binding protein that is known to

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A No coating Fibronectin 231 Exo 231 Nisch Exo C 400 231 Cells **** NC Fibronectin 300 231 Exosomes

200 231 Nisch **** Exosomes

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lls e 1 C h Cells 3 c B No coating Fibronectin 231 Exo 231 Nisch Exo 2 231 Nisch 231 Nis cells D. D 231 on Nisch exo 231 on NC 231 on Fb 231 on exo pPaxillin E No coating Fibronectin 231 Exo 231 Nisch Exo Paxillin 231 Cells Vinculin

G 150 G. NC **** Fibronectin 100 231 Exosomes **** 231 Nisch Exosomes

Area/1000 50

lls lls F No coating Fibronectin 231 Exo 231 Nisch Exo Ce Ce h 231 Nisc 1 231 Nisch 23

cells Aspect ratio 10 *** NC 8 Fibronectin **** 231 Exosomes 6 231 Nisch 4 Exosomes

0 s ls ll el e C C 1 3 ch 2 s

231 Ni

Figure 4. Exosomes from Nischarin cells reduce focal adhesions. A and B, Vinculin immunofluorescence of 231 cells on NC (n ¼ 9), fibronectin (n ¼ 9), 231 exosomes (n ¼ 10), or 231 Nisch exosomes (n ¼ 9; A) and 231 Nisch cells on NC (n ¼ 30), fibronectin (n ¼ 26), 231 exosomes (n ¼ 25), or 231 Nisch exosomes (n ¼ 30; inset with a zoom is shown; B). C, The number of focal adhesions (FA) per cell was determined by CellProfiler. D, Western blot analysis of pPaxillin, paxillin, and vincilin in 231 cells grown on NC, fibronectin, 231 exosomes, and 231 Nisch exosomes. E and F, Phalloidin immunofluorescence of 231 cells on NC (n ¼ 9), fibronectin (n ¼ 9), 231 exosomes (n ¼ 8), or 231 Nisch exosomes (n ¼ 11; E) and 231 Nisch cells on NC (n ¼ 23), fibronectin (n ¼ 25), 231 exosomes (n ¼ 25), or 231 Nisch exosomes (n ¼ 23; inset with a zoom is shown; F). G, Area and aspect ratio were acquired by ImageJ. Images were captured at 60 using a Nikon Eclipse Ti-S fluorescent microscope. Scale bars, 10 mm. , P < 0.0001. affect cell migration by antagonizing the actions of cell signaling also analyzed for focal adhesion dynamics. Consistent with 231 þ proteins that contribute to tumor cell migration and invasion (2). cells data, Nisch / cells had greater number of focal adhesions on þ þ The data in Fig. 4A–C is also consistent with previously published all coatings when compared with Nisch / cells (Supplementary data showing that Nischarin affects cytoskeletal reorganization, Fig. S3A and S3B). Increased number of focal adhesions were seen þ mainly by inhibiting Rac-induced lamellipodia formation (2). when cells plated on Nisch / exosomes. These results suggest To determine the effects of exosomes from Nischarin-reduced that there is a reduction in focal adhesion signaling when cells are cells biochemically, we seeded cells on different matrices, and attached to exosomes from Nischarin-positive cells and increased þ collected the lysates for protein detection. Paxillin is a focal signaling in Nisch / cells. adhesion scaffold protein and one of the first proteins to be Increased focal adhesion number often leads to increased cell recruited to focal adhesions during activation (32–36). We spreading. Thus, we wished to determine whether the increased observed an increase in paxillin expression when 231 cells were number of focal adhesions induced by exosomes from Nischarin- attached to 231 exosomes (Fig. 4D). Furthermore, the phosphor- reduced cells led to increased cell spreading. MDA-MB-231 and ylation of paxillin was visibly decreased in 231 cells on 231 Nisch 231 Nisch cells were seeded onto NC, fibronectin, 231 exosomes þ þ þ exosomes (Fig. 4D). Similarly, Nisch / and Nisch / cells were and 231 Nisch exosomes, and imaged. Visually, the cells appeared

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to be enlarged when seeded onto fibronectin and 231 exosomes still have an indirect effect that alters the sizes of exosomes. To (Fig. 4E and F). Using ImageJ, we analyzed the average area of each examine this, we isolated exosomes from 231 Rab14 cell lines. cell and found that the 231 cells had a greater average area on all S25N Rab14 overexpression significantly decreased exosome coatings compared with 231 Nisch cells (Fig. 4G). Among 231 diameter and molecular weight, while the constitutively active cells, the cell areas on fibronectin and 231 exosomes was similar, Rab14 has a significant increase (Fig. 5F), suggesting that the while the areas on control (NC) and 231 Nisch exosomes was presence of active Rab14 leads to increased exosome diameter almost identical (Fig. 4G). The differences in aspect ratio (cell and number (sample molecular weight). These results indicate length:breadth) between 231 and 231 Nisch cells seeded on 231 that Rab14 plays a significant role in exosome biogenesis and and 231 Nisch exosomes were significant. The area and aspect thus we propose that active Rab14 in 231 cells is responsible for ratio of 231 cells seeded on 231 Nisch exosomes are significantly the "oncogenic" exosomal phenotype. low compared with the cells seeded on 231 exosomes (Fig. 4G). This interaction between Nischarin and Rab14 has been well Similar results were obtained when the 231 Nisch cells seeded on established in our previous studies (13), but we have yet to 231 Nisch exosomes. These results indicate that 231 exosomes demonstrate the biological significance of these genes in breast enhance cell spreading. cancer. A study of human patients with breast cancer with variable Furthermore, we examined the effect of Nischarin-positive levels of Nisch determined that those with high Nisch have a exosomes on cell spreading using our mouse tumor cells. greater probability of relapse-free survival (Fig. 5G). In contrast, þ þ þ Nisch / and Nisch / cells were seeded onto NC, fibronectin, patients with lower RAB14 have a greater probability of relapse- þ þ þ Nisch / exosomes and Nisch / exosomes, and stained with free survival than those with high levels of RAB14 (Fig. 5H). These phalloidin. Our primary mouse tumor cells are significantly larger data agree with our previous experiments that showed an increase þ than the Nisch / cells, but their size expands even more when in the proliferation of Rab14-transfected cells (Fig. 5B). These þ they are on Nisch / exosomes (Supplementary Fig. S3C and studies demonstrate the importance of Nischarin and Rab14 in þ S3D). ImageJ analysis revealed an increased area of Nisch / cells breast cancer. þ þ compared with Nisch / cells on all coatings (Supplementary Fig. S3C and S3D). Furthermore, we noticed the greatest spreading of Exosomes from Nischarin cells reduce tumor volume þ þ Nisch / cells on fibronectin and Nisch / exosomes (Supple- Exosomes play an important role on tumor growth and thus it mentary Fig. S3C and S3D). These data reveal that attachment of is necessary to understand the affect that they have on tumors. To cancer cells to exosomes from Nischarin-reduced cells enhances explore the effects of Nischarin-reduced exosomes in vivo,we þ þ þ adhesion and cell spreading. isolated exosomes from Nisch / and Nisch / and cocultured þ þ þ them with Nisch / or Nisch / cells for 4 days, then injected the Active Rab14 is involved in exosomal trafficking cells into SCID mice (Fig. 6A). As a negative control, the exosomes Our data indicate that the exosomes from Nischarin-expressing alone were injected and, as a positive control, the cells alone cells are less effective than the exosomes from cells lacking (without coculturing) were injected. Tumor volumes were mea- Nischarin expression in promoting migration and spreading. sured every 3 days until the humane endpoint of the whole study. þ However, the underlying mechanism for this remains unknown. Nisch / exosomes significantly increased the tumor volume of þ þ þ þ Nischarin interacts with the trafficking GTPase Rab14 (13). On the Nisch / cells, while coculturing with Nisch / exosomes did not basis of Rab14's role in vesicle trafficking, we hypothesized that produce tumors by the end of the study (Fig. 6B). Concurrently, þ Nischarin's interaction with Rab14 determines the fate of exo- Nisch / exosomes also significantly increased the tumor volume þ þ þ þ somes. Our hypothesis is supported by previous evidence from of Nisch / cells, while Nisch / cells cocultured with Nisch / our laboratory demonstrating that Nischarin, Rab14, and the exosomes did not produce any tumors (Fig. 6C). This does not þ þ exosome marker CD63 colocalize (13). To determine whether suggest that Nisch / exosomes are not capable of stimulating this triple colocalization affects exosome trafficking, we created tumorigenesis, but they may do so at a delayed time compared þ Rab14 stably expressing MDA-MB-231 cell lines (Fig. 5A). A Q70L with Nisch / exosomes because the study had reached its Rab14 single point mutation yields a constitutively active protein, humane endpoint. At 44 days postinjection, we were able to þ while the S25N mutation yields a dominant-negative protein conclude that Nisch / exosomes increase the tumor volume of (37–39). First, we examined the role of Rab14 in cell proliferation both Nischarin-positive and reduced cells. We further isolated the using our novel cell lines. Introducing the dominant-negative tumors and confirmed this visually (Fig. 6D). To visualize the Rab14 did not have a significant effect on cell proliferation, while tumor architecture, we sectioned and stained the tumors with the WT and constitutively active Rab14 increased cell proliferation H&E (Fig. 6E). To confirm the number of proliferating cells, we þ þ þ (Fig. 5B). Very little is known about the role Rab14 plays in breast stained tissue sections with Ki67. Nisch / and Nisch / cells that þ cancer. To determine the further implications of Rab14 in breast were previously cocultured with Nisch / exosomes produced the cancer, we surveyed other breast cancer cell lines and found that greatest number of proliferative cells per area (Fig. 6F). Rab14 is expressed in MCF7, BT20, T47D, MDA-MB-468, and As reported above, our data showed that exosomes from þ SUM185 cells (Fig. 5C). Nisch / tumors have greater diameter and molecular weight Because we hypothesized that Rab14 contributes to exosome (Fig. 2E and F). A physiological consequence of this would be an trafficking, we assessed whether Rab14 is present in exosomes. increase in the overall stiffness of the tumor due to a greater Insignificant amounts of Rab14 RNA were found in the exo- exosomal burden. Changes in stiffness during cancer progression somes from the various 231 Rab14 cells, while the exosomal are important in understanding the pathophysiology of cancer RNA marker Rab27a was clearly present (Fig. 5D). Rab14 cells and metastatic mechanisms of cancer. Tumor progression is protein is also not expressed in exosomes (Fig. 5E) as indicated characterized by gradual stiffening of the tissue. We measured the þ þ þ by lysates that are positive for Rab27a (Supplementary Fig. stiffness of tumors from Nisch / and Nisch / animals using a S2B). Even though Rab14 is not present in exosomes, it may novel identification model system we developed (see Materials

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AB C DCells Exosomes 231 231 Rab14S25N 231 Rab14 231 Rab14Q70L MCF7 BT20 T47D MDA-MB-468 SUM 185 231 Rab14 231 231 Rab14S25N 231 231 Rab14 231 Rab14Q70L 231 Rab14Q70L Rab14 Rab14 231 Rab14S25N Rab27a Vinculin Vinculin Rab14 GAPDH

EFDiameter Molecular weight 150 4 * * * * 3 100

2 (/1 E 08) 231 Cells 231 Exo 231 Nisch Exo 231 Nisch Cells 50

Diameter (nm) 1

Rab14 Molecular weight

0 0

Vinculin N N L FP 5 0L FP 5 G 2 1 G S Q70 3 31 4 2 2 1 14 b14 S2 b14 Q7 b b a Ra Ra Ra R P FP FP F FP G G 1 31 G 3 31 2 231 G 2 2

G H

NISCH Relapse-free survival RAB14 Relapse-free survival

Figure 5. Active Rab14 is involved in the intracellular trafficking of exosomes. A, Western blot detection of Rab14 and vinculin (control) in 231, 231 Rab14 S25N, 231Rab14Q70L,and231Rab14WTcells.B, Cell proliferation of 231, 231 Rab14 S25N, 231 Rab14 Q70L, 231 Rab14 WT, and 231 Nisch cells by MTT (n ¼ 5each).C, Western blot detection of Rab14 and vinculin (control) in MCF7, BT20, T47D, MDA-MB-468, and SUM185 cells. D, RT-PCR detection of Rab27a, Rab14, and GAPDH in 231, 231 Rab14 S25N, 231 Rab14 Q70L, 231 Rab14 WT cells (left) and exosomes (right). E, Western blot detection of Rab14 and vinculin (control) in protein lysates and exosomes from 231 and 231 Nisch cells. F, Diameter and molecular weight analysis of exosomes from 231, 231 Rab14 S25N, 231 Rab14 Q70L, and 231 Rab14 WT with the DelsaNanoC. G and H, Relapse-free survival of human patients with high and low expression of NISCHARIN (G)andRAB14 (H). , P < 0.05; , P < 0.001. and Methods for details). We compared the mechanical properties Talin-1 reduces cell stiffness, which suggests that focal adhesion of both types of tumors by measuring Young's moduli. We found signaling increases cell stiffness. In this study, for the first time, þ that Nisch / animals produce stiffer tumors (Fig. 6G). We we show that Nischarin-negative tumors are much stiffer than propose that this increased stiffness is partly due to the exosomes. Nischarin-positive tumors. An important step in metastasis formation is cancer cell invasion through tissues. It has been shown that metastatic cells indent Nischarin-reduced cells confer resistance to cell-cycle control stiff polyacrylamide gels to promote invasion, whereas benign by exosomes cells do not indent (40), suggesting that this approach can be used So far, we have shown that coculturing cells with Nischarin- for tumors to assess their potential metastatic potential. The positive exosomes significantly delays tumor growth. To deter- mechanical properties of cancer cells change depending on the mine whether these Nischarin-positive exosomes increase apo- stiffness of the ECM. This stiffness depends on stable focal ptosis, we performed apoptosis assays on the cocultured cells adhesion proteins, such as Talin 1. For example, suppression of from Fig. 6 prior to their injection into the mice. By staining the

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A B C 100 1,200 *** +/+ +/-

) *** ) Nisch Exosomes

Nisch Exosomes 3 3 *** *** 80 Nisch+/+ Cells *** Nisch+/- Cells +/- +/+ ** Nisch+/+ Cells w/ Nisch+/+ *** *** Nisch Cells w/ Nisch *** *** *** *** 60 Exosomes *** *** Exosomes Nisch+/+ Cells w/ 600 *** Nisch+/- Cells w/ Nisch+/- 40 *** ** ** Nisch+/+ Nisch+/- Nisch+/- Exosomes *** *** *** Exosomes * Cells Cells 20 * Tumor volume (mm Tumor volume (mm * *** *** 0 0 6 2 7 0 2 5 17 2 y 20 y 23 y y 35 y 41 y1 y2 y 23 y3 y3 y 38 ay a a a ay 3 a ay 38 a a a D D D D Day 29D D D D Da Da D Day 26Day 29Da Da D Day 41 D E

+/+ +/- +/+ Nisch+/- Nisch Nisch Nisch Cells Cells Cells Cells

Nisch+/+ Cells Nisch+/- Cells 100 µm 100 µm +/- +/- w/ Nisch w/ Nisch +/- Nisch+/+ Cells Nisch+/- Nisch+/+ Nisch Exosomes Exosomes Cells w/ +/- Cells w/ Cells w/ +/- w/ Nisch +/- Nisch +/- Nisch Exosomes Nisch Exosomes Exosomes Exosomes 100 µm 100 µm

*** F *** G +/- Tumor stiffness Nisch+/+ Nisch 500 ** 20 Cells Cells 400 * 15 300

100 µm 100 µm 200 10 Cells per area KPa + Nisch+/- 100

Cells w/ #Ki67 5 Nisch+/- 0 s lls o os Exosomes ells x 0 - /+ Ce / C /- Ex /- E + + + + h h h T 100 µm 100 µm ch c c M s isc Ni Nis + - Py / -PyMT +/ - w/ N w/ Nis + +/+ +/- h ch is sc Nisch Nisch Ni N

Figure 6. Exosomes from Nischarin cells reduce tumor volume. A, Prkdcscid mice injected with Nischþ/þ or Nischþ/ cells, and Nischþ/þ or Nischþ/ cells previously cocultured with Nischþ/ exosomes. B, Tumor volumes of mice injected with Nischþ/þ (n ¼ 3) exosomes, Nischþ/þ (n ¼ 3) cells, or Nischþ/þ cells previously cocultured with Nischþ/þ (n ¼ 3) or Nischþ/ (n ¼ 3) exosomes. C, Tumor volumes of cells injected with Nischþ/ (n ¼ 3) exosomes, Nischþ/ (n ¼ 4) cells, or Nischþ/ cells previously cocultured with Nischþ/þ (n ¼ 4) or Nischþ/ exosomes (n ¼ 4). Red asterisks indicate statistical signiﬁcance to the exosome groups. Black asterisks indicate statistical signiﬁcance between the cells only group. D, Isolated mammary tumors from mice injected with Nischþ/þ or Nischþ/ cells, and Nischþ/þ or Nischþ/ cells previously cocultured with Nischþ/ exosomes. E, H&E staining of tumors. F, Ki67 staining of tumors with quantitation of the number of Ki67-positive cells per area (inset with a zoom is shown). G, Tumor stiffness (in kPa) of Nischþ/þ and Nischþ/ tumors. , P < 0.05; , P < 0.01; , P < 0.001.

cells with Annexin and propidium iodide and counting with data in Fig. 6B that show an increase in tumor cell growth of þ þ þ flow cytometry, we were able to determine the percentage of Nisch / cells cocultured with Nisch / exosomes. Overall, þ þ þ þ necrotic and apoptotic cells. Coculturing Nisch / cells with these data show that Nisch / exosomes increase apoptosis in þ þ þ Nisch / exosomes decreased the percentage of live cells both cell lines, while Nisch / exosomes do not induce þ þ (Fig. 7A). For the Nisch / cells, the percentage of cells in apoptosis. þ apoptosis (early and late apoptosis) rose from 5.4% in control The apoptosis experiments with Nisch / cells corroborated þ þ þ conditions, to 10.8% after coculturing with the Nisch / exo- the data in Fig. 6C. For the Nisch / cells, total apoptosis somes (Fig. 7A). These findings might explain why there was a increased from 1.1% to 20.1% after coculturing with the þ þ þ þ decrease in the tumor growth of the Nisch / cells cocultured Nisch / exosomes, also explaining why we saw delayed growth þ þ þ þ with Nisch / exosomes. In contrast, coculturing Nisch / cells at the humane endpoint of the study (Fig. 7B). Furthermore, when þ þ þ with Nisch / exosomes decreased the percentage of cells in the Nisch / cells were cocultured with the Nisch / exosomes, þ þ apoptosis to 5.3%, which was lower than Nisch / cells the percentage of live cells returned to 97.9%, which was the same þ þ þ (Fig. 7A). This was further confirmed by caspase-3 staining of as the Nisch / control (Fig. 7B). As with the Nisch / cells, þ þ þ þ þ the mouse tumors from Fig. 6D. Nisch / cells cocultured with Nisch / exosomes increased apoptosis, while Nisch / exosomes þ Nisch / exosomes had the greatest amount of caspase-3 stain- had the opposite effect. We next assessed cell proliferation using a þ þ ing, while Nisch / cells cocultured with Nisch / exosomes different approach by performing a MTT assay on the cells cocul- þ þ had the least amount of caspase-3–positive cells (Supplemen- tured with Nisch / exosomes prior to injection into the SCID þ þ þ þ tary Fig. S4A and S4B). The data in Fig. 7A also match the mice. Coculturing Nisch / cells with Nisch / exosomes slightly

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+/+ +/+ +/- +/- Nisch+/- Cells ABNisch+/+ Cells Nisch Cells w/ Nisch Cells Nisch Cells Nisch Cells w/ Nisch+/+ Exosomes w/ Nisch+/- Exosomes Nisch+/+ Exosomes w/ Nisch+/- Exosomes

2.4% 15.3% 2.4% 4.3% 1.0% 4.5% 1.3% 2.3% 0.9% 0.5% 0.5% 0.5%

97.9% 0.6% 77.5% 4.8% 91.4% 1.1% 88.2% 6.3% 93.5% 3.0% 97.9% 1.1%

MDA-MB-231 20 CD*** No Exosomes EG1 (49%)S (42%) G2–M (8%) G1 (65%)S (26%) G2–M (8%) +/+ 15 *** Nisch Exosomes Nisch+/- Exosomes

10 *** GFP GFP-Nisch 5 Cyclin D1 Relative absorbance 0 Vinculin lls lls Ce /- Ce +/+ + ch MDA-MB231-GFP MDA-MB231-GFP-Nisch isch is N N

Nisch+/+ Cells Nisch+/+ Cells +/+ +/- Nisch+/+ Cells w/ Nisch w/ Nisch F Exosomes Exosomes G1 (32.2%) G1 (65.4%) G1 (42.1%) S (29%) S (21.1%) S (44.3%) G2 (22.5%) G2 (22.1%) G2 (15.3%)

Figure 7. Nisch+/- Cells Nisch+/- Cells Nischarin-reduced cells confer resistance to cell-cycle control by exosomes. þ/þ +/+ +/- A and B, Annexin/propidium iodide apoptosis assay of Nisch and cells +/- w/ Nisch w/ Nisch Nisch Cells þ/þ þ/ G Exosomes Exosomes previously cocultured with no exosomes, and Nisch or Nisch exosomes (A) and Nischþ/ cells previously cocultured with no exosomes, G1 (23.4%) G1 (85%) G1 (24.2%) þ/þ þ/ þ/þ S (34.7%) S (14.6%) S (33.7%) and Nisch or Nisch exosomes (B). C, MTT assay of Nisch and Nischþ/ cells previously cocultured with no exosomes, Nischþ/þ exosomes, G2 (16.5%) G (21.9%) G (21%) 2 2 or Nischþ/ exosomes. D, Western blot analysis of cyclin D1 and vinculin in 231 and 231 Nisch cells. E-G, Flow cytometric analysis of the cell cycle with propidium iodide in 231 and 231 Nisch cells (E), as well as Nischþ/þ (F) and Nischþ/ cells with Nischþ/þ exosomes or Nischþ/ exosomes (G). , P < 0.001.

reduced cell proliferation, while culturing the same cells with thepercentageofcellsinG1 phase from 32.2% to 65.4%, while þ Nisch / exosomes increased proliferation (Fig. 7C). Interestingly, decreasing the percentage of cells in S-phase from 29% to þ þ þ þ þ coculturing Nisch / cells with Nisch / exosomes still increased 21.1% (Fig. 7F). Furthermore, coculturing Nisch / cells with þ þ proliferation, while culturing them with Nisch / exosomes Nisch / exosomes increases the percentage of cells in S-phase increased proliferation even more (Fig. 7C). These results further from 29% in controls to 44.3% (Fig. 7F). These results þ þ þ þ þ confirmed that Nisch / exosomes increase cell proliferation. suggest that coculturing Nisch / cells with Nisch / exosomes þ/ þ/ Although Nisch exosomes increase cell proliferation, increased G1 cell-cycle arrest, while coculturing with Nisch þ þ Nisch / exosomes slightly decrease WT cell proliferation. We exosomes with other types of cells enhances progression then determined whether these exosomes are halting cell-cycle through S-phase. þ þ control. Nischarin decreases the expression of cyclin D1 Furthermore, our mouse tumor cells showed 29% of Nisch / þ/ (Fig. 7D), a regulator of G1–S-phase progression (41). Further cells in S-phase compared with 34.7% of Nisch cells (Fig. 7G). þ þ þ FACS analysis of propidium iodide–stained cells showed that After coculturing Nisch / cells with Nisch / exosomes, the 231 Nisch cells have an increase in G1 cell-cycle arrest, which number of cells in G1 phase increased from 23.4% to 85% increased from 49% in 231 cells to 65% in 231 Nisch cells (Fig. 7G). This significant increase may explain why these cells (Fig. 7E). While this confirms that Nischarin regulates the cell had not formed tumors at the endpoint of the study. Furthermore, þ þ cycle, we wanted to determine whether Nischarin-reduced or coculturing Nisch / cells with Nisch / exosomes yielded positive exosomes also contribute to cell-cycle regulation. To percentages of cells in G1 and S-phase close to control determine whether there was cell-cycle arrest after coculturing cells (Fig. 7G). Taken together, our cell-cycle data show that þ þ þ þ þ with the exosomes, we performed cell-cycle analysis again. coculturing Nisch / and Nisch / cells with Nisch / exosomes þ/þ þ/þ Coculturing Nisch cells with Nisch exosomes increased induces G1 cell-cycle arrest.

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Discussion exosomes have an important effect on focal adhesion signaling Our data demonstrate that exosome production is decreased in through their effect on paxillin phosphorylation. Furthermore, we þ Nischarin-expressing cells, while Nisch / cells enhance the pro- propose that Nischarin's interaction with active Rab14 regulates duction of exosomes. Exosomes from Nischarin-positive cells exosome production, which in turn affects cell adhesion, cell promote migration and in vivo tumor growth of orthotopic breast migration, tumor growth, and metastasis. In contrast, exosomes cancer cells. Although Nischarin functions as a tumor suppressor, from cells with reduced levels of Nischarin expression may migrate its role in exosome release is unknown. Exosomes are released by and establish a metastatic niche, which favors enhanced metastatic cancer cells and are known to promote cancer progression. In this growth. In conclusion, our work shows that the secretion of article, we report that the decrease of Nischarin expression aug- exosomes by breast tumors in vivo can regulate tumor progression. ments exosome shedding. Coculturing of na€ve breast cancer cells In addition, Nischarin-expressing tumor-derived exosomes þ with exosomes secreted by Nischarin-reduced (Nisch / ) cells decrease tumor progression. These data may or may not be promote focal adhesion formation, cell migration, tumor growth, applicable to all tumors as the idiosyncrasies of each tumor vary. and metastasis. Colocalization of Rab14 with Nischarin appears In this study, we characterized the effects of Nischarin on cell to be important for the regulation of exosome production and motility. In the presence of Nischarin, there is a triple co-local- release. In patients with breast cancer, Rab14 mRNA overexpres- ization between Nischarin, Rab14, and CD63 that reduces the sion in the primary tumor is associated with decreased overall release of exosomes (Supplementary Fig. S5). Furthermore, cells survival and, in an orthotopic mouse model, Rab14 inhibition that are seeded on exosomes from Nischarin-reduced cells have impairs breast cancer progression. increased phosphorylation of the focal adhesion scaffold protein, We have demonstrated that human breast cancer cells that paxillin. Our findings identified the presence of HSP70, Flot1, express Nischarin have reduced cell proliferation. We suggest that RAB27A, ITGA5, ITGA11, ITGAL, and ITGAV mRNA in exosomes. this might be due to insufficient cell attachment. This hypothesis The cargo present in exosomes is poorly characterized; however, was also based on previous findings that linked Nischarin to our results contribute significantly to the understanding of exo- decreasing the function of key cell attachment proteins, such as some biology. An additional contribution of our findings is that FAK (1, 42). Also, it has been shown by other investigators that exosomes have the ability to transform cells into highly metastatic Nischarin regulates apoptosis and metastasis of breast cancer (43), agents. This novel role for the tumor suppressor Nischarin loss of Nischarin promotes cell proliferation metastasis of ovarian not only increases our understanding of exosome biology, cancer (42), and suppression of Nischarin promotes neuronal but can be translated to identifying new targets for modulating migration (44), suggesting that Nischarin has significant role in cancer metastasis. other cancers as well. We found that Nischarin-reduced cells had increased proliferation regardless of the substrate they were seed- Disclosure of Potential Conflicts of Interest ed on. Nischarin is one of many tumor suppressors whose down- No potential conflicts of interest were disclosed. regulation leads to cancer cell death resistance. PTEN and Tropo- myosin-1 are examples of tumor suppressors that induce cancer cell death upon expression in breast cancer cells and primary Authors' Contributions breast tumors (45, 46). When a cell is undergoing cell death, it Conception and design: M. Maziveyi, S. Baranwal, S.K. Alahari fi Development of methodology: M. Maziveyi, S. Dong, S. Baranwal, S.K. Alahari detaches from the ECM. We also con rmed that Nischarin Acquisition of data (provided animals, acquired and managed patients, decreases cell spreading and focal adhesion number. This is a provided facilities, etc.): M. Maziveyi, S. Dong, T.M. Huckaba, K. Park novel role for Nischarin in regulating the number of adhesion Analysis and interpretation of data (e.g., statistical analysis, biostatistics, points between a cell and its external matrix. computational analysis): M. Maziveyi, R. Ratthinam, T.M. Huckaba, We showed that Nischarin alters the size of exosomes and D.E. Mercante, S.K. Alahari further explored the implications of these changes. We first Writing, review, and/or revision of the manuscript: M. Maziveyi, A. Mehrnezhad, T.M. Huckaba, S.K. Alahari assessed the migration of cells on exosomes. We previously pub- Administrative, technical, or material support (i.e., reporting or organizing lished that Nischarin reduces cell migration of 231 cells when data, constructing databases): M. Maziveyi, S. Dong, A. Mehrnezhad, stably transfected (4). Fibronectin itself promotes cell migration in R. Ratthinam, S.K. Alahari many cell models (47–49). In fact, increased total fibronectin Study supervision: M. Maziveyi, S.K. Alahari expression correlates with poorer prognosis in patients with cancer (50–52). Coating tissue culture dishes with fibronectin signif- Acknowledgments icantly increased the maximum distance and mean velocity. Some- We would like to thank the laboratory of Dr. Tarun Mandal at Xavier times it increased these parameters to a greater extent than the University of Louisiana for allowing us to use the Malvern Nanosight and the Beckman DelsaNanoC. Also, we thank the Fred Brazda Foundation and LSU exosomes did. Nischarin-reduced human and mouse exosomes School of Medicine for financial support. promoted a greater distance and velocity than Nischarin-positive D.E. Mercante was supported in part by U54 GM104940 from the National exosomes. Because cancer cell migration is generally regarded as a Institute of General Medical Sciences of the National Institutes of Health, which prerequisite for metastasis, we propose a novel mechanism by funds the Louisiana Clinical and Translational Science Center. The content is which Nischarin exerts its tumor-suppressive functions. solely the responsibility of the authors and does not necessarily represent the fi Exosomes have been shown to promote the invasion and of cial views of the National Institutes of Health. migration of cancer cells. Also, multiple studies indicated that The costs of publication of this article were defrayed in part by the payment of exosomes migrate and localize to future sites of metastasis, page charges. This article must therefore be hereby marked advertisement in which will establish a metastatic niche into which cancer cells will accordance with 18 U.S.C. Section 1734 solely to indicate this fact. spread. Our results demonstrate that exosomes promote or inhibit metastasis through their migration regulation depending on Received March 20, 2018; revised October 19, 2018; accepted January 8, 2019; the Nischarin expression levels. This study demonstrates that published first January 11, 2019.

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Nischarin Regulates Exosome Production

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2166 Cancer Res; 79(9) May 1, 2019 Cancer Research

Exosomes from Nischarin-Expressing Cells Reduce Breast Cancer Cell Motility and Tumor Growth

Mazvita Maziveyi, Shengli Dong, Somesh Baranwal, et al.

Cancer Res 2019;79:2152-2166. Published OnlineFirst January 11, 2019.

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