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

Author Manuscript Published OnlineFirst on January 11, 2019; DOI: 10.1158/0008-5472.CAN-18-0842 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Exosomes from Nischarin-Expressing Cells Reduce Breast Cancer Cell Motility and tumor growth

Mazvita Maziveyi1,7, Shengli Dong1, Somesh Baranwal2, Ali Mehrnezhad3, Rajamani Rathinam4, Thomas M. Huckaba5, Donald E. Mercante6, Kidong Park3, Suresh K. Alahari1

1Department of Biochemistry and Molecular Biology, LSUHSC School of Medicine, New Orleans, LA, USA 2Center of Biochemistry and Microbial Science, Central University of Punjab, Bathinda-151001, India 3Department of Electrical Engineering and Computer Engineering, Louisiana State University, Baton Rouge, LA, USA 4Wayne State University, Detroit, MI, USA 5Department of Biology, Xavier University of Louisiana, New Orleans, LA, USA 6School of Public Health, LSUHSC School of Medicine, New Orleans, LA, USA 7Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas

*Corresponding author; Suresh K. Alahari, PhD; Fred G. Brazda Professor of Biochemistry, LSUHSC School of Medicine, New Orleans, LA 70112, USA; Tel: 504-568-4734 [email protected]

Running Title: Nischarin regulates exosome production

Conflicts of Interest

No potential conflicts of interest were disclosed.

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 11, 2019; DOI: 10.1158/0008-5472.CAN-18-0842 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract:

Exosomes are small extracellular microvesicles that are secreted by cells when intracellular multivesicular bodies (MVB) fuse with the plasma membrane. We have previously demonstrated that Nischarin inhibits focal adhesion formation, cell migration, and invasion, leading to reduced activation focal adhesion kinase. In this study, we propose that the tumor suppressor Nischarin regulates the release of exosomes. When co-cultured on exosomes from Nischarin-positive cells, breast cancer cells exhibited reduced survival, migration, adhesion, and spreading. The same co-cultures formed xenograft tumors of significantly reduced volume following injection into mice. Exosomes secreted by Nischarin-expressing tumors inhibited tumor growth. Expression of only one allele of Nischarin increased secretion of exosomes, and Rab14 activity modulated exosome secretions and cell growth. Taken together, the present study reveals a novel role for Nischarin in preventing cancer cell motility, which contributes to our understanding of exosome biology.

Significance: Regulation of Nischarin-mediated exosome secretion by Rab14 seems to play an important role in controlling tumor growth and migration.

Introduction:

Nischarin, or imidazoline receptor antisera-selected protein (IRAS), is a protein involved in a number of biological processes. The Nisch gene is located on chromosome 3p21, which is frequently lost in cancers (1). Most notably, Nischarin is an Integrin α5β1 binding protein known to affect cell migration by antagonizing the actions of cell signaling proteins that contribute to tumor cell migration and invasion (2). Furthermore, Nischarin has also been shown to affect cytoskeletal reorganization, mainly by inhibiting Rac-induced lamellipodia formation (2). Consistent with this, Nischarin’s inhibition of cell migration has been linked to other proteins (3- 5).

During cell migration, cells adhere to its extracellular environment through focal adhesions (FAs). These complexes use Integrins to attach to extracellular matrix (ECM) proteins (6,7). Each Integrin has designated ligand(s), and decreased expression of the ligand or receptor affects FA number. Integrins also bind to Fibronectin-coated exosomes (8). Exosomes are smaller microvesicles (30-200 nm in diameter) secreted from cells when multivesicular bodies (MVBs) fuse with the plasma membrane (9-12). Although Nischarin’s role has yet to be linked to exosomes, previous studies have shown that the Nischarin-Rab14 interaction promotes the maturation of CD63 positive endosomes (13). Nischarin is an effector of the GTPase Ras- related protein Rab-14 (13). Although Rab14 is involved in vesicle sorting and trafficking (14), only one report has identified Rab14 function in breast cancer exosomes (15). Nischarin directly interacts with Rab14 to effect intracellular Salmonella survival (13). In the presence of Nischarin, there is triple co-localization between the late endosome and exosome marker CD63, Rab14, and Nischarin (13).

While it is known that MVBs fuse with the plasma membrane just before exosomes release, the physiological consequences of this have yet to be determined in the breast cancer microenvironment. Furthermore, the proteins responsible for the MVB-plasma membrane fusion are not well characterized. We hypothesized that Nischarin may affect the migration of cancer cells by controlling exosome release. Exosomes from 231 cells promoted migration of 231 cells, while exosomes from 231 Nisch cells inhibited migration. These effects were due to the decreased number of exosomes released by 231 Nisch cells. In contrast, active Rab14 promotes exosome secretion and cell growth. In summary, our study highlights the anti-tumoral function of Nischarin expression mediated by exosome-dependent secretions in breast cancer.

Materials and Methods

Cell Culture

All cells (MDA-MB-231, MDA-MB-231 Nischarin, MCF10A, MCF7, BT20, T47D, MDA-MB-468, SUM185 and MCF7) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) at 37°C, 5%

CO2 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco, Waltham, MA). MDA-MB-231 Nisch, MDA-MB-231 GFP, MDA-MB-231 Rab14, MDA-MB-231 Rab14 S25N, and MDA-MB-231 Rab14 Q70L were prepared as previously described (4). Briefly, 231 Nisch cells were generated by amplifying human NISCH. The 4545 base pair PCR product was then cloned into a 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 particles were collected 48 hours later, concentrated, then reconstituted in serum-free media. The MDA-MB-231 GFP cells were generated similarly, except there was no cloning of NISCH into the pCDH-CMV-MCS-EF1-copGFP vector. The cells were sorted by FACS for GFP selection. Low passage cells were used for all the experiments, and the cell line purify was verified every two months using appropriate markers of the cell type. Transfected cells were selected using antibiotic puromcyin.

Nischarin WT (+/+), Nischarin HET (+/-) and Nischarin Null (-/-) animals were generated as described before (16). Briefly, exons 7 to 10 of Nischarin were deleted, and the resulting animals were intercrossed with animals expressing the mouse mammary tumor virus-polyma middle T transgene. For mouse genotyping, mouse tail genomic DNA was extracted and amplified by PCR and electrophoresed on 2% agarose gels. Primary WT-PyMT (Nisch+/+), HET- PyMT (Nisch+/-) and Null-PyMT (Nisch -/-) cells were isolated as previously described (17). Briefly, the mammary tumors were isolated and cut into small pieces with a razor blade and scissors. The tissues were incubated with collagenase for two hours to allow for enzymatic dissociation of the tissue. The resulting material was ultra-centrifuged to remove debris and blood. The following conditions were used for cell culture experiments of cells that were seeded on ECM proteins, 10ug/ml of Fibronectin (BD Biosciences, San Diego, CA) was prepared in PBS. Bovine Collagen 1 ((BD Biosciences, San Diego, CA) was added to each well at 0.16ml/cm2. The ECM proteins were added to the wells and placed on a rocker for 2 hours at room temperature then washed two times with warm PBS. The cells were seeded onto the wells immediately after washing with PBS.

Cell line authentication: MDA-MB-231 cells were obtained from ATCC and Nischarin expression in these cells was maintained by puromycin selection. Nischarin expression was monitored by immunoblotting using anti-Nischarin antibody. Cells were not used beyond passage five and mycoplasma was tested for all cell lines at least once every six months. The primary cells prepared from PyMT tumors were tested every time for Nischarin truncation genotype, PyMT expression by genomic PCR approach. The primary cells were never used beyond passage three.

Animal Studies

All mouse experiments were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the Louisiana State University Health Sciences Center, New Orleans. Four- to ten-week-old female Prkdc scid mice were used in the xenograft studies (4 mice per group). The exosomes used for all mouse co-injections were isolated from 1.8g of mouse mammary tumor. Therefore, the approximate number of Nisch+/+ exosomes isolated were 1 x 108 while the number of Nisch+/- exosomes was 1.9 x 108. After exosome isolation, animals were either injected with exosomes only, 1 x 106 viable Nisch+/+ or Nisch+/- cells alone, or Nisch+/+ or Nisch+/- cells that were incubated with the Nisch+/+ or Nisch+/- exosomes for 4 days. Tumor growth was assessed every three days with calipers and tumor volume was calculated as π x length x width2/6.

Isolation of Exosomes

3 million cells were seeded in T175 flasks and cultured for 48 hours. The media were centrifuged at 300 x g for 10 minutes, and then the supernatant was collected and centrifuged again at 2,000 x g for 20 minutes. This was repeated again for two more centrifugations both at 10,000 x g for 30 minutes and 100,000 x g for 70 minutes. The supernatant was then discarded and the pellet was centrifuged with PBS for 70 minutes at 100,000 x g. The pellet was finally resuspended in PBS for subsequent studies. To isolate exosomes from whole tumors, we harvested breast tumors from Nisch+/+ and Nisch+/- mice as previously described (17). Tumors were disnintegrated and incubated with collagenase for 3 hours. The samples were centrifuged for 5 minutes at 200 x g. The supernatant was then used for the exosome isolation procedure by ultracentrifugation.

The Beckman Couter DelsaTMNanoC instrument was used to measure diameter and molecular weight. The samples were diluted in ddH2O, added to a cuvette, and inserted into the device. This instrument uses Photon Correlation Spectroscopy (PCS) to analyze the molecular weight of the sample as it relates to the α and β ion particles in the solution. Basically, when laser light is directed towards the particle, light will scatter in different directions. The intensities observed by the machine are a result of the movement of ion particles due to Brownian motion. For particle counting, the Malvern NanoSight, a Nanoparticle Tracking Device (NTA) instrument was used. The samples were diluted in ddH2O and added to the flow system with a syringe. As the samples pass through the flow system, the exosomes are visualized, counted and characterized.

Labeling of Exosomes

Exosomes were isolated from Nisch+/+ and Nisch+/- tumors and re-suspended in PBS. For CD63 labelling, the CD63 antibody was added at a concentration of 1:100 and incubated for 30 minutes at 37°C as previously described (18). The exosomes were rinsed in 0.5% BSA-PBS and centrifuged at 100,000 x g for 1 hour at 4°C. The secondary antibody (Alexa Fluor 594) was added at a 1:200 concentration for 30 minutes at 37°C. The exosomes were washed again, and the pellet was re-suspended with PBS. Exosome images were captured using the Leica DMi8 confocal microscope.

Antibodies

Antibodies and dilutions were used as follows: mouse monoclonal anti-Vinculin (Sigma, St. Louis, MO; 1:5000), mouse monoclonal anti-Paxillin (BD Biosciences, San Diego, CA; 1:5000), rabbit monoclonal anti-Flotillin (Cell Signaling, Danvers, MA; 1:100-500), mouse monoclonal anti-CD63 (Santa Cruz, Dallas, TX; 1:500), mouse monoclonal anti-Rab14 (Santa Cruz, Dallas, TX; 1:100-1000), mouse monoclonal anti-Nischarin (BD Biosciences, San Diego, CA; 1:1000), and Phalloidin (Sigma, St. Louis, MO; 1:100).

Cell Proliferation Assay

For MTT cell viability assays, cells were seeded onto 96-well plates for 24-48 hours with the various coatings. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent

(5mg/ml in PBS) was added to each well. After a 3.5 hour incubation, the media/MTT was aspirated and a HCl/isopropanol solution (0.1% NP-40) was added to each well. The plate was incubated for 20 minutes and then the absorbance was measured at 565nm. The results were normalized to medium alone control.

For cell counting experiments, 5,000 231 GFP, 231 GFP Nisch, Nisch+/+, Nisch+/- cells were seeded onto 96 well dishes and incubated for 24 hours. After 24 hours, representative images were captured at 10x with the EVOS XL Cell Imaging System. The number of cells per milliliter were calculated using the Bio-Rad TC20 Automated Cell Counter.

Live Cell Migration

For live cell imaging, the cells were seeded onto 12 well plates and cultured at 37 °C with 5% CO2 overnight. The next day, a small wound was created on the corner of the well with a 10 µl pipette tip to stimulate migration, and live cell migration was captured every hour for 19 hours. Max distance and mean velocity were calculated using the ImageJ plugins MTrackJ and Chemotaxis Tool. Migration tracks were created by using the X and Y coordinates from the MTrackJ plugin. These coordinates were tracked by the microscope and registered at subpixel precision as the inverse of the zooming factor. For data representation, the subsequent X and Y points were subtracted from the previous X and Y points. The represented axis boundaries were selected based on the cell that traveled the most distance and standardized for each cell type. For example, the 231 cell that traveled the furthest distance on the –X axis reached (-313.333,- 97.333), therefore the –X axis for all 231 cells was set to -350m. Images were captured at 10X using the Olympus IX81 and Nikon Eclipse Ti-S.

Immunofluorescence

Cells were washed two times with PBS and fixed in 3.7% formaldehyde for ten minutes. Then, the cells were washed three times with PBS for five minutes each. The cells were then permeabilized with 0.5% Triton-X100 for five minutes. The cells were washed again three times with PBS then blocked for one hour with 2% BSA (bovine serum albumin) in PBS. After blocking, the coverslips were incubated upside down on the primary antibody overnight (Vinculin or CD63) at 4°C. After primary antibody staining, the cells were washed three times for ten minutes each. The cells were then incubated with the secondary antibody for one hour. Prior to mounting, the cells were washed twice with PBS for ten minutes, then once with water. The coverslips were mounted with Fluoromount-G (Southern Biotech, Birmingham, AL).

For Phalloidin staining, cells were washed two times with PBS, and fixed in 3.7% formaldehyde for five minutes. The cells were washed three times with PBS for five minutes each and were stained with 50µg/ml of a fluorescently conjugated Phalloidin (Sigma, St. Louis, MO) for thirty minutes. After staining, the cells were washed three times in PBS for ten minutes each. The coverslips were mounted with DAPI Fluoromount-G (Southern Biotech, Birmingham, AL).

CellProfiler was used to determine the number of focal adhesions per cell and the percentage of area covered by focal adhesions. After incubating the cells overnight on Fibronectin, the cells were fixed and stained with Vinculin. Images of Vinculin-stained cells were uploaded into

CellProfiler and the Enhance or Suppress Features and Indentify Primary Objects modules were used to assess the focal adhesions. For the Identify Primary Objects module, the typical diameter used was 10-50. The threshold strategy was Global, the thresholding method was Robust Background. The lower bounds on threshold was then changed to 0.001.Images were captured at 60X using the Nikon Eclipse Ti-S.

Real-time PCR.

Total RNA was isolated from cultured cells with TRIzol reagent (Invitrogen, Carlsband, CA). cDNA was generated from 2ug of RNA using the Invitrogen/Applied Biosystems/ABl High Capacity cDNA Reverse Transcriptase Kit (Carlsband, CA). For gel detection, samples were run on a 3% agarose gel.

Stiffness with Indentation

A cylindrical sample with 6 mm diameter was cut out from the tumor tissue and constrained by a PDMS mold. The sample is mounted inside a petri dish on top of an electronic scale (SPX622, Ohaus, USA), which is located on a motorized stage (H101E1F, PRIOR scientific, USA). The motorized stage brings the sample up and pushes it through a fixed circular indenter with 1 mm diameter, which produces a known surface displacement of the sample. The resulting force is measured simultaneously using the scale and the force versus strain curve is extracted. To calculate the stiffness, an indentation model is used considering the geometry of the indenter (19). The stage is lifted with step size of 10 μm until a force of 5 mN is applied on the sample. To avoid the adhesion effect (20), the average slope over the region between 50% and 100% of the maximum indentation force is defined as contact stiffness, k. The effective elastic modulus,

Eeff can be defined using the standard relation (21) as shown in Equation 1. ì ü p k ï E = ï ï eff 2 A ï k í ý Þ Eeff = Equation (1) ï pd 2 ï d ï A = ï î 4 þ where, A is the projected contact area underneath the indenter and d is the diameter of the circular indenter.

Generally the effective elastic modulus is a function of elastic modulus of both indenter and sample. However since the elasticity of used indenter, Ei is much larger than the elasticity of the sample, Es, this relation can be simplified as shown in Equation 2

ì 2 2 ü 1 1-n 1-n ï = s + i ï ï ï 2 í Eeff Es Ei ý Þ Es = (1-n s )Eeff Equation (2) ï ï E >> E îï i s þï

where, νi and νs are Poisson’s ratio of the indenter and the sample. Assuming that the sample is incompressible (νs = 0.5), the elastic modulus of the sample is extracted from contact stiffness as shown in Equation (3). 3k E = Equation (3) s 4d

Statistical Analysis

All experiments were repeated at least three times. Statistical analyses involving only one factor were performed using Graphpad Prism 5.0 software (San Diego, CA) using either a two-tailed t test or one-way ANOVA. Experiments involving two factors were analyzed in SAS (Version 9.4) with two-factor ANOVA with interaction. Pairwise mean comparisons were performed using the PDIFF option on the LSMEANS statement of PROC GLM in SAS. The error bars indicate the standard deviation from the mean. *p<0.05 **p<0.01 and ***p<0.001 and ****p<0.0001. All experiments were repeated at least three times.

Results:

Nischarin Regulates Cell Attachment.

Detachment of cells from a matrix leads to anoikis, cell death due to the loss of adhesion (22,23). Since Nischarin reduces the activation of proteins, such as FAK and Rac that contribute to cell adhesion, we examined whether there was a decrease in cell attachment in Nischarin- positive breast cancer cells seeded on different matrices. To perform this, we used our previously published MDA-MB-231 cells stably expressing Nischarin (hereafter, 231 Nisch) and Nisch+/+-Polyoma Middle T (PyMT), Nisch+/-- Polyoma Middle T (PyMT) and Nisch-/-- Polyoma Middle T (PyMT) cells (4). To determine whether Nischarin affects cell proliferation, we seeded MDA-MB-231 (hereafter, 231) and 231 Nisch cells on 2D (directly on the tissue culture plate), Fibronectin and Collagen and measured cell proliferation using the MTT assay and automated cell counting. The human breast cancer cells stably expressing Nischarin had a decrease in cell proliferation on 2D, Fibronectin and Collagen (Fig 1A and Supp Fig 1A). Similarly, we checked cell proliferation of Nisch+/+-PyMT, Nisch+/--PyMT and Nisch-/--PyMT mouse tumor cells on 2D, Fibronectin, and Collagen. Hereafter these tumor cells are referred to as Nisch+/+, Nisch+/- and Nisch-/- cells. On 2D, only Nisch-/- had a significant increase in proliferation. On Fibronectin and Collagen, Nisch+/- cells had significantly greater proliferation compared to Nisch+/+ (Fig 1B and Supp Fig 1B). Overall, Nisch-/- cells had the greatest amount of proliferation compared to

Nisch+/+ and Nisch+/- cells. In all of the cell lines we tested, the presence of Nischarin in cancer cells led to a significant reduction in cell proliferation regardless of the matrix (Fig 1A-B).

There are many possible contributors to reduced cell proliferation. To determine whether the reduction in cell proliferation of Nischarin-positive breast cancer cells was due to cell attachment, we assessed cell spreading and focal adhesion dynamics. 231 and 231 Nisch cells were seeded onto Fibronectin, stained with Phalloidin and captured by immunofluorescence microscopy. Cell area and aspect ratio were quantified with ImageJ. Nischarin decreased the average cell area and aspect ratio (Fig 1C). This “shrinking” of cells is an indicator of cancer cell death. To determine whether this reduction of cell spreading is due to a decrease in FAs, we stained cells with the FA marker vinculin, then determined the number of FAs and the area covered by FAs using CellProfiler. 231 Nisch cells had a reduction in FA number and in the area covered by FAs (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 Nischarin regulates attachment to specific components of the ECM remains elusive.

Nischarin Alters Exosomes Properties.

The ECM is a diverse mesh network of proteins that support cell attachment. The ECM stimulates PI3K activity, which results in cancer phenotypes, such as anchorage independent growth on soft agar and protection from apoptosis (24,25). Previous reports show that culturing non-cancerous cells in conditioned media from cancer cells promotes proliferation and migration of non-cancerous cells (26,27). We previously determined that incubating non-cancerous MCF10A cells with media from 231 cells promotes cell migration (28). Incubating the cells with media from 231 Nisch cells promotes a moderate amount of cell migration, but significantly less than media from 231 cells (28). These results suggest that Nischarin is regulating the cancer cell secretome. The possible factors involved in this regulation could be matrix metalloproteases, cytokines, or secreted exosomes, to name a few. Thus, we first examined whether Nischarin is regulating the release of exosomes.

Although each cell type secretes unique exosomes, CD63 and Flotillin1 are commonly used exosomal markers for mammary cells. Flotillin proteins function in a number of contexts, such as endocytosis and cell signaling. We isolated exosomes from 231 and 231 Nisch cells by differential ultracentrifugation. We validated Flotillin1 expression in exosomes and cells by Western Blotting (Fig 2A). Previous studies have shown that Flotillin1 is expressed in whole cell lysates, as well as, in exosomes from 231 cells (29). RNAs are also present in exosomes (30). We observed the expression of HSP70, ITGA5, ITGA11, ITGAL, and ITGAV mRNA in the exosomes (Fig 2B). This further confirms that we have isolated exosomes. Very little amounts of Nisch mRNA and protein were found in the exosomes (Fig 2B and Supp Fig 2A) and Rab27a is not detected in exosomes (Supp Fig 2B).

We examined the mean diameter of our exosomes (9-12). Photon Correlation Spectroscopy (PCS) indicated that our exosomes are between 30-200nm. Though not statistically significant, exosomes from 231 cells had a mean diameter of 189nm while those from 231 Nisch cells have a lower mean diameter of 172nm (Fig 2C). Since only small variations were observed in vesicle size between 231 and 231 Nisch samples, we next assessed changes in exosome quantity.

Using PCS, we were able to analyze the molecular weight of the sample as it relates to α and β ion particles in the solution. Although not statistically significant, exosomes from 231 cells had a molecular weight of 1.49x109 while those from 231 Nisch cells had a weight of 1.24x109 (Fig 2C). Since we did not see significant differences in the diameter and molecular weight of 231 and 231 Nisch cells, we assessed particle number using the Nanoparticle Tracking Analysis (NTA) instrument (Malvern NanoSight). 231 Nisch cells had a decreased number of particles per frame (counted by the software) and per milliliter (Fig 2D). These results suggest that Nischarin-positive cells produce less exosomes than Nischarin-reduced cells. Furthermore, we performed rescue experiments in Nisch knockdown cells to see whether we can reduce particle counts. To do this, we introduced Nisch into previously published MCF7 cells (4) which have a stable knockdown of Nisch. Introducing Nisch to knockdown cells reduced exosome numbers to control levels (Supp Fig 2C, 2D). These data indicate that Nischarin-positive 231 cells secrete significantly fewer exosomes while Nischarin-positive MCF7 cells secrete slightly fewer exosomes when compared to their Nisch-negative counterparts.

We also examined exosome number using whole mouse tumor tissues. Exosomes secreted from Nisch+/+ and Nisch+/- tumors are also CD63 positive (Fig 2E-F). We assessed the diameter and molecular weight of exosomes. Nisch+/+ tumor exosomes had an average diameter of 67nm, which was significantly lower than the Nisch+/- exosome size (98nm) (Fig 2G). Furthermore, we found that for the same sized tumor, Nisch+/- exosomes had an average weight of 3.99x108 while Nisch+/+ exosome samples had a significantly lower weight (1.86x108) (Fig 2G). Finally, Nisch+/- and Nisch-/- exosomes have a slightly greater number of particles per frame, as well as number of particles per ml, although not statistically significant (Fig 2H). Taken together, our results suggest that the presence of Nischarin in cells promotes secretion of fewer and smaller exosomes compared to cells that have non-functional Nischarin.

Exosomes from Nischarin-Positive Cells Reduce Breast Cancer Cell Motility.

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

greater distance and velocity on Fibronectin, followed by on 231 exosomes, then on 231 Nisch exosomes.

Next, we examined migration using mouse tumor exosomes from animals with different Nischarin profiles. We investigated whether the altered exosome characteristics from Nischarin- positive cells affects cell motility in mouse tumors. Fewer Nisch-/- animals are viable; therefore, in order to maintain significant replicates for our experiments, we only used Nisch+/+ and Nisch+/- tumor cells. Nisch+/+ or Nisch+/- tumor cells were seeded on tissue culture dishes coated with no substrate, Fibronectin, exosomes from Nisch+/+ tumors, or exosomes from Nisch+/- tumors and migration was tracked every hour for 19 hours. The migration was most dramatic for Nisch+/+ and Nisch+/- cells on Nisch+/- exosomes (Fig 3D,E). When mean velocity was assessed, there was a slight increase in the migration of Nisch+/- cells on fibronectin , although not statistically significant. Furthermore, Nisch+/+ cells migration on Nisch+/- exosomes is significantly higher than on Nisch+/+ exosomes (Fig 3F). Nisch+/- exosomes promoted greater cell velocity compared to Fibronectin and Nisch+/+ exosome coating. The most significant changes between Nisch+/+ and Nisch+/- cells were seen with the exosome coatings (3F). Generally, exosomes from Nischarin-reduced cells increased cell migration compared to Nischarin-positive cells. The data suggests that the absence of Nischarin may lead the cells to increase release of exosomes for an added migratory advantage.

Exosomes from Nischarin-Positive Cells Reduce Cell Adhesion.

From the above data, we have established a potential role for exosomes in cell migration. Cells seeded on Fibronectin have greater dynamic adhesions and cell spreading. The presence of dynamic adhesions determines that whether a cell focal adhesion machinery is able to disassemble and assemble in a highly motile cell. To understand how these exosomes reduce cell migration, we examined cell adhesion to the exosomes and changes in FA numbers. MDA- MB-231 and 231 Nisch cells were seeded onto NC, Fibronectin, 231 exosomes and 231 Nisch exosomes, and visualized the FA complexes. In addition, we analyzed the number of FAs per cell using CellProfiler. MDA-MB-231 cells displayed an increased number of focal adhesions on all matrices, while the FAs on 231 exosomes showed further increase in number. However, FAs on 231 Nisch cells are significantly lower in number (Fig 4A-C). Furthermore, 231 cells plated on 231 Nisch exosomes showed less number of FAs. As stated before Nischarin is an Integrin α5β1 binding protein that is known to affect cell migration by antagonizing the actions of cell signaling proteins that contribute to tumor cell migration and invasion (2). The data in Fig 4A-C is also consistent with previously published data showing that Nischarin affects cytoskeletal reorganization, mainly by inhibiting Rac-induced lamellipodia formation (2).

To determine the effects of exosomes from Nischarin-reduced cells biochemically, we seeded cells on different matrices, and collected the lysates for protein detection. Paxillin is a FA scaffold protein and one of the first proteins to be recruited to FAs during activation (32-36). We observed an increase in Paxillin expression when 231 cells were attached to 231 exosomes (Fig 4D). Furthermore, the phosphorylation of Paxillin was visibly decreased in 231 cells on 231 Nisch exosomes (Fig 4D). Similarly, Nisch+/+ and Nisch+/- cells were also analyzed for FA dynamics. Consistent with 231 cells data, Nisch+/- cells had greater number of FAs on all

coatings when compared to Nisch+/+ cells (Supp Fig 3A, 3B). Increased number of FAs were seen when cells plated on Nisch+/- exosomes. These results suggest that there is a reduction in FA signaling when cells are attached to exosomes from Nischarin-positive cells and increased signaling in Nisch+/- cells.

Increased FA number often leads to increased cell spreading. Thus, we wished to determine whether the increased number of FAs induced by exosomes from Nischarin-reduced cells led to increased cell spreading. MDA-MB-231 and 231 Nisch cells were seeded onto NC, Fibronectin, 231 exosomes and 231 Nisch exosomes and imaged. Visually, the cells appeared to be enlarged when seeded onto Fibronectin and 231 exosomes (Fig 4E-F). Using ImageJ, we analyzed the average area of each cell and found that the 231 cells had a greater average area on all coatings compared to 231 Nisch cells (Fig 4G). Among 231 cells, the cell areas on Fibronectin and 231 exosomes was similar, while the areas on control (NC) and 231 Nisch exosomes was almost identical (Fig 4G). The differences in aspect ratio (cell length: breadth) between 231 and 231 Nisch cells plated on 231 and 231 Nisch exosomes were significant. The area and aspect ratio of 231 cells plated on 231 Nisch exosomes are significantly low compared to the cells plated on 231 exosomes (Fig 4G). Similar results were obtained when the 231 Nisch cells plated on 231 Nisch exosomes. These results indicate that 231 exosomes enhance cell spreading.

Furthermore, we examined the effect of Nischarin-positive exosomes on cell spreading using our mouse tumor cells. Nisch+/+ and Nisch+/- cells were seeded onto NC, Fibronectin, Nisch+/+ exosomes and Nisch+/- exosomes, and stained with Phalloidin. Our primary mouse tumor cells are significantly larger than the Nisch+/- cells, but their size expands even more when they are on Nisch+/- exosomes (Supp Fig 3C, D). ImageJ analysis revealed an increased area of Nisch+/- cells compared to Nisch+/+ cells on all coatings (Supp Fig 3C, D). Furthermore, we noticed the greatest spreading of Nisch+/- cells on Fibronectin and Nisch+/- exosomes (Supp Fig 3C, D). These data reveal that attachment of cancer cells to exosomes from Nischarin-reduced cells enhances adhesion and cell spreading.

Active Rab14 is Involved in Exosomal Trafficking

Our data indicate that the exosomes from Nischarin expressing cells are less effective than the exosomes from cells lacking Nischarin expression in promoting migration and spreading. However, the underlying mechanism for this remains unknown. Nischarin interacts with the trafficking GTPase Rab14 (13). Based on Rab14’s role in vesicle trafficking, we hypothesized that Nischarin’s interaction with Rab14 determines the fate of exosomes. Our hypothesis is supported by previous evidence from our lab demonstrating that Nischarin, Rab14 and the exosome marker CD63 co-localize (13). To determine whether this triple co-localization affects exosome trafficking, we created Rab14 stably expressing MDA-MB-231 cell lines (Fig 5A). A Q70L Rab14 single point mutation yields a constitutively active protein, while the S25N mutation yields a dominant negative protein (37-39). First, we examined the role of Rab14 in cell proliferation using our novel cell lines. Introducing the dominant negative Rab14 did not have significant effect on cell proliferation, while the WT and constitutively active Rab14 increased cell proliferation (Fig 5B). Very little is known about the role Rab14 plays in breast cancer. To

determine the further implications of Rab14 in breast cancer, we surveyed other breast cancer cell lines and found that Rab14 is expressed in MCF7, BT20, T47D, MDA-MB-468, and SUM185 cells (Fig 5C).

Since we hypothesized that Rab14 contributes to exosome trafficking, we assessed whether Rab14 is present in exosomes. Insignificant amounts of Rab14 RNA were found in the exosomes from the various 231 Rab14 cells, while the exosomal RNA marker Rab27a was clearly present (Fig 5D). Rab14 protein is also not expressed in exosomes (Fig 5E) as indicated by lysates that are positive for Rab27a (Supp Fig 2B). Even though Rab14 is not present in exosomes, it may still have an indirect effect to alter the sizes of exosomes. To examine this, we isolated exosomes from 231 Rab14 cell lines. S25N Rab14 overexpression significantly decreased exosome diameter and molecular weight, while the constitutively active Rab14 has a significant increase (Fig 5F) suggesting that the presence of active Rab14 leads to increased exosome diameter and number (sample molecular weight). These results indicate that Rab14 plays a significant role in exosome biogenesis and thus we propose that active Rab14 in 231 cells is responsible for the “oncogenic” exosomal phenotype.

This interaction between Nischarin and Rab14 has been well established in our previous studies (13), but we have yet to demonstrate the biological significance of these genes in breast cancer. A study of human breast cancer patients with variable levels of Nisch determined that those with high Nisch have a greater probability of relapse-free survival (Fig 5G). In contrast, patients with lower RAB14 have a greater probability of relapse-free survival than those with high levels of RAB14) (Fig 5H). These data agree with our previous experiments that showed an increase in the proliferation of Rab14 transfected cells (Fig 5B). These studies demonstrate the importance of Nischarin and Rab14 in breast cancer.

Exosomes from Nischarin Cells Reduce Tumor Volume

Exosomes play an important role on tumor growth and thus it is necessary to understand the affect that they have on tumors. To explore the effects of Nischarin-reduced exosomes in vivo, we isolated exosomes from Nisch+/+ and Nisch+/- and incubated them with Nisch+/+ or Nisch+/- cells for 4 days, then injected the cells into SCID mice (Fig 6A). As a negative control, the exosomes alone were injected and, as a positive control, the cells alone were injected. Tumor volumes were measured every 3 days until the humane endpoint of the whole study. Nisch+/- exosomes significantly increased the tumor volume of Nisch+/+ cells, while incubation with Nisch+/+ exosomes did not produce tumors by the end of the study (Fig 6B). Concurrently, Nisch+/- exosomes also significantly increased the tumor volume of Nisch+/- cells, while Nisch+/- cells incubated with Nisch+/+ exosomes did not produce any tumors (Fig 6C). This does not suggest that Nisch+/+ exosomes are not capable of stimulating tumorigenesis, but they may do so at a delayed time compared to Nisch+/- exosomes since the study had reached its humane endpoint. At 44 days post-injection, we were able to conclude that Nisch+/- exosomes increase the tumor volume of both Nischarin-positive and reduced cells We further isolated the tumors and confirmed this visually (Fig 6D). To visualize the tumor architecture, we sectioned and stained the tumors with H&E (Fig 6E). To confirm the number of proliferating cells, we stained

tissue sections with Ki67. Nisch+/+ and Nisch+/- cells that were previously co-cultured with Nisch+/- exosomes produced the greatest number of proliferative cells per area (Fig 6F).

As reported above, our data showed that exosomes from Nisch+/- tumors have greater diameter and molecular weight (Fig 2E-F). A physiological consequence of this would be an increase in the overall stiffness of the tumor due to a greater exosomal burden. Changes in stiffness during cancer progression are important in understanding the pathophysiology of cancer cells and metastatic mechanisms of cancer. Tumor progression is characterized by gradual stiffening of the tissue. We measured the stiffness of tumors from Nisch+/+ and Nisch+/- animals using a novel identification model system we developed (see Materials and Methods for details). We compared the mechanical properties of both types of tumors by measuring with Young’s moduli. We found that Nisch+/- animals produce stiffer tumors (Fig 6G). We propose that this increased stiffness is partly due to the exosomes. An important step in metastasis formation is cancer cell invasion through tissues. It has been shown that metastatic cells indent stiff polyacrylamide gels to promote invasion, whereas benign cells do not indent (40) suggesting that this approach can be used for tumors to assess their potential metastatic potential. The mechanical properties of cancer cells change depending on the stiffness of the ECM. This stiffness depends on stable FA proteins, such as Talin 1. For example, suppression of Talin-1 reduces cell stiffness, which suggests that FA signaling increases cell stiffness. In this study for the first time we show that Nischarin-negative tumors are much stiffer than Nischarin-positive tumors.

Nischarin-Reduced Cells Confer Resistance to Cell Cycle Control by Exosomes

So far, we have shown that co-culturing cells with Nischarin-positive exosomes significantly delays tumor growth. To determine whether these Nischarin-positive exosomes increase apoptosis, we performed apoptosis assays on the co-cultured cells from Fig 6 prior to their injection into the mice. By staining the cells with Annexin and propidium iodide and counting with flow cytometry, we were able to determine the percentage of necrotic and apoptotic cells. Incubating Nisch+/+ cells with Nisch+/+ exosomes decreased the percentage of live cells (Fig 7A). For the Nisch+/+ cells, the percentage of cells in apoptosis (early and late apoptosis) rose from 5.4% in control conditions, to 10.8% after co-culturing with the Nisch+/+ exosomes (Fig 7A). These findings might explain why there was a decrease in the tumor growth of the Nisch+/+ cells incubated with Nisch+/+ exosomes. In contrast, co-culturing Nisch+/+ cells with Nisch+/- exosomes decreased the percentage of cells in apoptosis to 5.3%, which was lower than Nisch+/+ cells (Fig 7A). This was further confirmed by Caspase 3 staining of the mouse tumors from Fig 6D. Nisch+/+ cells co-cultured with Nisch+/- exosomes had the greatest amount of Caspase 3 staining, while Nisch+/- cells co-cultured with Nisch+/- exosomes had the least amount of Caspase 3 positive cells (Supp Fig 4 A, B). The data in Figure 7A also match the data in Figure 6B that shows an increase in tumor cell growth of Nisch+/+ cells incubated with Nisch+/- exosomes. Overall, these data show that Nisch+/+ exosomes increase apoptosis in both cell lines, while Nisch+/- exosomes do not induce apoptosis.

The apoptosis experiments with Nisch+/- cells corroborated the data in Figure 6C. For the Nisch+/- cells, total apoptosis increased from 1.1% to 20.1% after co-culturing with the Nisch+/+ exosomes, also explaining why we saw delayed growth at the humane endpoint of the study

(Fig 7B). Furthermore, when the Nisch+/- cells were co-cultured with the Nisch+/- exosomes, the percentage of live cells returned to 97.9%, which was the same as the Nisch+/- control (Fig 7B). As with the Nisch+/+ cells, Nisch+/+ exosomes increased apoptosis, while Nisch+/- exosomes had the opposite effect. We next assessed cell proliferation using a different approach by performing a MTT assay on the cells co-cultured with Nisch+/+ exosomes prior to injection into the SCID mice. Co-culturing Nisch+/+ cells with Nisch+/+ exosomes slightly reduced cell proliferation, while culturing the same cells with Nisch+/- exosomes increased proliferation (Fig 7C). Interestingly, co-culturing Nisch+/- cells with Nisch+/+ exosomes still increased proliferation, while culturing them with Nisch+/- exosomes increased proliferation even more (Fig 7C). These results further confirmed that Nisch+/- exosomes increase cell proliferation.

Although Nisch+/- exosomes increase cell proliferation, Nisch+/+ exosomes slightly decrease WT cell proliferation. We then determined whether these exosomes are halting cell cycle control. Nischarin decreases the expression of Cyclin D1 (Fig 7D), a regulator of G1 to S phase progression (41). Further FACS analysis of propidium iodide stained cells showed that 231 Nisch cells have an increase in G1 cell cycle arrest, which increased from 49% in 231 cells to 65% in 231 Nisch cells (Fig 7E). While this confirms that Nischarin regulates the cell cycle, we wanted to determine whether Nischarin-reduced or positive exosomes also contribute to cell cycle regulation. To determine whether there was cell cycle arrest after co-culturing with the exosomes, we performed cell cycle analysis again. Co-culturing Nisch+/+ cells with Nisch+/+ exosomes increased the percentage of cells in G1 phase from 32.2% to 65.4%, while decreasing the percentage of cells in S phase from 29% to 21.1% (Fig 7F). Furthermore, co- culturing Nisch+/+ cells with Nisch+/- exosomes increases the percentage of cells in S phase from 29% in controls to 44.3% (Fig 7F). These results suggest that co-culturing Nisch+/+ cells with Nisch+/+ exosomes increased G1 cell cycle arrest, while co-culturing with Nisch+/- exosomes with other types of cells enhances progression through S phase.

Furthermore, our mouse tumor cells showed 29% of Nisch+/+ cells in S phase compared to 34.7% of Nisch+/- cells (Fig 7G). After co-culturing Nisch+/- cells with Nisch+/+ exosomes, the number of cells in G1 phase increased from 23.4% to 85% (Fig 7G). This significant increase may explain why these cells had not formed tumors at the endpoint of the study. Furthermore, co-culturing Nisch+/- cells with Nisch+/- exosomes yielded percentages of cells in G1 and S phase close to control cells (Fig 7G). Taken together, our cell cycle data shows that co-culturing Nisch+/+ and Nisch+/- cells with Nisch+/+ exosomes induces G1 cell cycle arrest.

Discussion:

Our data demonstrate that exosome production is decreased in Nischarin-expressing cells, while Nisch+/- cells enhance the production of exosomes. Nischarin exosomes promote migration and in vivo tumor growth of orthotopic breast cancer cells. Although Nischarin functions as a tumor suppressor, its role in exosome release is unknown. Exosomes are released by cancer cells and are known to promote cancer progression. In this paper, we report that the decrease of Nischarin expression augments exosome shedding. Co-culturing of naïve breast cancer cells with exosomes secreted by Nischarin-reduced (Nisch+/-) cells promote FA formation, cell migration, tumor growth and metastasis. Co-localization of Rab14 with Nischarin

appears to be important for the regulation of exosome production and release. In breast cancer patients, Rab14 mRNA overexpression in the primary tumor is associated with decreased overall survival and in an orthotopic mouse model Rab14 inhibition impairs breast cancer progression.

We have demonstrated that human breast cancer cells that express Nischarin have reduced cell proliferation. We suggest that this might be due to insufficient cell attachment. This hypothesis was also based on previous findings that linked Nischarin to decreasing the function of key cell attachment proteins, such as FAK (1,42). Also, it has been shown by other investigators that Nischarin regulates apoptosis and metastasis of breast cancer (43), loss of Nischarin promotes cell proliferation metastasis of ovarian cancer (42), and suppression of Nischarin promotes neuronal migration (44) suggesting that Nischarin has significant role in other cancers as well. We found that Nischarin-reduced cells had increased proliferation regardless of the substrate which they were seeded on. Nischarin is one of many tumor suppressors whose down regulation leads to cancer cell death resistance. PTEN and Tropomyosin-1 are examples of tumor suppressors that induce cancer cell death upon expression in breast cancer cells and primary breast tumors (45,46). When a cell is undergoing cell death, it detaches from the ECM. We also confirmed that Nischarin decreases cell spreading, and FA number. This is a novel role for Nischarin in regulating the number of adhesion points between a cell and its external matrix.

We showed that Nischarin alters the size of exosomes and further explored the implications of these changes. We first assessed the migration of cells on exosomes. We previously published that Nischarin reduces cell migration of 231 cells when stably transfected (4). Fibronectin itself promotes cell migration in many cell models (47-49). In fact, increased total Fibronectin expression correlates with poorer prognosis in cancer patients (50-52). Coating dishes with Fibronectin significantly increased the maximum distance and mean velocity. Sometimes it increased these parameters to a greater extent than the exosomes did. Nischarin-reduced human and mouse exosomes promoted a greater distance and velocity than Nischarin-positive exosomes. Since cancer cell migration is generally regarded as a prerequisite for metastasis, we propose a novel mechanism by which Nischarin exerts its tumor suppressive functions.

Exosomes have been shown to promote the invasion and migration of cancer cells. Also, multiple studies indicated that exosomes migrate and localize to future sites of metastasis which will establish a metastatic niche into which cancer cells will spread. Our results demonstrate that exosomes promote or inhibit metastasis through their migration regulation depending on the Nischarin expression levels. The present study demonstrates that exosomes have an important effect on FA signaling through their effect on Paxillin phosphorylation. Furthermore, we propose that Nischarin’s interaction with active Rab14 regulates exosome production, which in turn affects cell adhesion, cell migration, tumor growth and metastasis. In contrast, exosomes with reduced levels of Nischarin expression may migrate and establish a metastatic niche, which favors enhanced metastatic growth. In conclusion, our work shows that the secretion of exosomes by breast tumors in vivo can regulate tumor progression. In addition, Nischarin- expressing tumor derived exosomes decrease tumor progression. These data may or may not be applicable to all tumors as the idiosyncrasies of each tumor vary.

In this study, we characterized the effects of Nischarin on cell motility. In the presence of Nischarin, there is a triple co-localization between Nischarin, Rab14 and CD63 that reduces the release of exosomes (Supp Fig 5). Furthermore, cells that are seeded on exosomes from Nischarin-reduced cells have up regulation of the FA scaffold protein, Paxillin Our findings identified the presence of HSP70, Flot1, RAB27A, ITGA5, ITGA11, ITGAL, and ITGAV mRNA in exosomes. The cargo present in exosomes is poorly characterized; however, our results contribute significantly to the understanding of exosome biology. An additional contribution of our findings is that exosomes have the ability to transform cells into highly metastatic agents. This novel role for the tumor suppressor Nischarin not only increases our understanding of the exosome biology, but can be translated to identifying new targets for modulating cancer metastasis.

Acknowledgements

We would like to thank the laboratory of Dr. Tarun Mandal at Xavier University of New Orleans for allowing us to use the Malvern Nanosight and the Beckman DelsaNanoC. Also, we thank the Fred Brazda Foundation and LSU School of Medicine for financial support.

References

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Figure Legends:

Figure 1. Nischarin Decreases the Attachment of Breast Cancer Cells. A) 231 (n=6) and 231 Nisch (n=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) Nisch+/+, Nisch+/-, and Nisch-/- cells were seeded onto a tissue culture surface (2D), Fibronectin or Collagen for 48 hours. Cell proliferation was determined by the MTT assay. C) 231 (n=18) and 231 Nisch (n=21) cells were seeded on 10µg/ml Fibronectin for 24 hours and stained with Phalloidin. The average area and aspect ratio were calculated using Image (inset with a zoom is shown) J. Aspect ratio= major axis/minor axis. D) Vinculin immunofluorescence staining of 231 and 231 Nisch cells (inset with a zoom is shown). Number of Vinculin positive FAs per area and % area covered by FA was calculated using CellProfiler. *p<0.05 **p<0.01 and ***p<0.001.

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, and ITGAV 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=9) and 231 Nisch (n=9) cells with the Delsa Nano C. D) Number of particles per frame and per ml of 231 (n=6) and 231 Nisch exosomes (n=15) with the Nanosight NTA. E) Nisch+/+ and Nisch+/- exosomes were labeled with CD63 (inset with a zoom is shown). Scale bars indicate 10µm. F) Western blot detection of CD63 and Vinculin (control) in Nisch+/+ tumor exosomes, Nisch+/+ tumors, Nisch+/- tumor +/- exosomes and Nisch tumor cells. G) Diameter and molecular weight (Mw) analysis of exosomes from Nisch+/+ (n=3) and Nisch+/- (n=3) tumors with the Delsa Nano C. H) Number of particles per frame and per mililiter of Nisch+/+ (n=6), Nisch+/- exosomes (n=11) and Nisch-/- exosomes (n=3) with the Nanosight NTA. *p<0.05 **p<0.01 and ***p<0.001.

Figure 3. Exosomes from Nischarin Cells Reduce Cell Motility. A) 231 and B) 231 Nisch cells were seeded onto NC, Fibronectin, 231 exosomes or 231 Nisch exosomes and live imaging was captured every hour for 19 hours (n=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 Nisch+/+ cells seeded on NC (n=4), Fibronectin (n=7), Nisch+/+ exosomes (n=11) or Nisch+/- exosomes (n=9). E) Migration tracks of Nisch+/- cells were seeded on NC (n=14), Fibronectin (n=19), Nisch+/+ exosomes (n=27) or Nisch+/- exosomes (n=10). F) Mean velocity of Nisch+/+ and Nisch+/- cells on NC, Fibronectin, Nisch+/+ exosomes and Nisch+/- exosomes. *p>0.05 **p<0.01 and ***p<0.001.

Figure 4. Exosomes from Nischarin Cells Reduce Focal Adhesions. A) Vinculin immunofluorescence of 231 cells on NC (n=9), Fibronectin (n=9), 231 exosomes (n=10) or 231 Nisch exosomes (n=9) and B) 231 Nisch cells on NC (n=30), Fibronectin (n=26), 231 exosomes

(n=25) or 231 Nisch exosomes (n=30) (inset with a zoom is shown). C) The number of FA’s per cell was determined by CellProfiler. D) Western blot of pPaxillin, Paxillin and Vincilin in 231 cells grown on NC, Fibronectin, 231 exosomes and 231 Nisch exosomes. E) Phalloidin immunofluorescence of 231 cells on NC (n=9), Fibronectin (n=9), 231 exosomes (n=8) or 231 Nisch exosomes (n=11) and (F) 231 Nisch cells on NC (n=23), Fibronectin (n=25), 231 exosomes (n=25) or 231 Nisch exosomes (n=23) (inset with a zoom is shown). G) Area and aspect ratio were acquired by ImageJ. Images were captured at 60X using a Nikon Eclipse Ti-S fluorescent microscope. Scale bars indicate 10µm. *p<0.05 **p<0.01 ***p<0.001 and ****p<0.0001.

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, 231 Rab14 Q70L, 231 Rab14 WT cells. B) Cell Proliferation of 231, 231 Rab14 S25N, 231 Rab14 Q70L, 231 Rab14 WT, and 231 Nisch cells by MTT (n=5 each). 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, 231 Rab14 WT with the Delsa Nano C. G) Relapse free survival of human patients with high and low expression of NISCHARIN and H) RAB14. *p<0.05 **p<0.01 and ***p<0.001.

Figure 6. Exosomes from Nischarin Cells Reduce Tumor Volume. A) Prkdcscid mice injected with Nisch+/+ or Nisch+/- cells, and Nisch+/+ or Nisch+/- cells injected with Nisch+/- exosomes. B) Tumor volumes of cells injected with Nisch+/+ (n=3) exosomes, Nisch+/+ (n=3) cells, or Nisch+/+ cells previously co-cultured with Nisch+/+ (n=3) or Nisch+/- (n=3) exosomes. C) Tumor volumes of cells injected with Nisch+/- (n=3) exosomes, Nisch+/- (n=4) cells, or Nisch+/- cells previously co- cultured with Nisch+/+ (n=4) or Nisch+/- exosomes (n=4). *Red asterisks indicate statistical significance to the exosome groups. *Black asterisks indicate statistical significance between to the cells only group. D) Isolated mammary tumors from mice injected with Nisch+/+ or Nisch+/- cells, and Nisch+/+ or Nisch+/- cells injected with Nisch+/- 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 Nisch+/+ and Nisch+/- tumors. *p<0.05 **p<0.01 and ***p<0.001.

Figure 7. Nischarin-Reduced Cells Confer Resistance to Cell Cycle Control by Exosomes. A) Annexin/Propidium Iodide apoptosis assay of Nisch+/+ and cells previously co-cultured with no exosomes, and Nisch+/+ or Nisch+/- exosomes and B) Nisch+/- cells previously co-cultured with no exosomes, and Nisch+/+ or Nisch+/- exosomes. C) MTT assay of Nisch+/+ and Nisch+/- cells previously incubated with no exosomes, Nisch+/+ exosomes, or Nisch+/- exosomes. D) Western blot of Cyclin D1 and Vinculin in 231 and 231 Nisch cells. E) Flow cytometric analysis of the cell cycle with propidium iodide in 231 and 231 Nisch cells, as well as, F) Nisch+/+ and G) Nisch+/- cells with Nisch+/+ exosomes or Nisch+/- exosomes. *p<0.05 **p<0.01 and ***p<0.001.

Figure 1: Nischarin Decreases the Attachment of Breast Cancer Cells

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 562nm Absorbance at 562nm Absorbance at 562nm Absorbance 0.0 0.0 0.0

MDA-MB-231 MDA-MB-231 MDA-MB-231

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Absorbance at 562nm Absorbance Absorbance at 562nm Absorbance Absorbance at 562nm Absorbance 0 0 0

-PyMT -PyMT -/- -PyMT + -PyMT -PyMT -/- -PyMT +/+ +/- -PyMT -PyMT -PyMT + +/- +/+ +/- -/- / Nisch Nisch Nisch Nisch Nisch Nisch Nisch Nisch Nisch MDA-MB-231 C. MDA-MB-231 MDA-MB-231 Nisch D. MDA-MB-231 Nisch

Average Area 4 800 4 150000 3 600 3 * * 100000 2 400 2 **

Area ** #FA per Area #FA

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N is c h - /- -P y M T Author Manuscript Published OnlineFirst on January 11, 2019; DOI: 10.1158/0008-5472.CAN-18-0842 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 3: Exosomes From Nischarin Cells Reduce Cell Motility

A. 231 Cells on NC 231 Cells on 231 Cells on 231 231 Cells on 231 250 Fibronectin Exosomes Nisch Exosomes C. 250 250 M a x D is ta n c e 4 0 0 150 N C

F ib ro n e c tin 3 0 0 -350 150 -50 2 3 1 E xo so m e s -350 150 -350 150 -350 150

m 2 0 0 * * 2 3 1 N is c h E x o s o m e s  -250 -250 -250 -250 1 0 0 B. 231 Nisch Cells on 231 Nisch Cells on 231 Nisch Cells on 231 Nisch Cells on 0 s s ll ll NC Fibronectin 231 Exosomes 231 Nisch e e C C 1 h 3 c Exosomes 2 is 250 N 250 250 1 3 250 2 M e a n V e lo c ity

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n * * -250 -250 -250 -250 i

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Exosomes s s 250 Exosomes ll ll 250 e e C C 1 h 3 c 250 250 2 is N 1 3 F. 2 -350 150 -350 150 -350 150 -350 150 M e a n V e lo c ity -250 -250 -250 -250 0 .2 0 * * * N C +/- +/- +/- F ib ro n e c tin +/- Nisch Cells on Nisch Cells on Nisch Cells on 0 .1 5 Nisch Cells on NC * * + / +

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Figure 4: Exosomes From Nischarin Cells Reduce Focal Adhesions A. No Coating Fibronectin 231 Exo 231 Nisch Exo 4 0 0 C. * * * * N C 231 cells F ib ro n e c tin

l 3 0 0 l

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A. Figure 6: Exosomes From Nischarin Cells Reduce Tumor Volume B. C. 100 1200 *** +/+ +/-

) ***

) 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 Volume Tumor Tumor Volume (mm Volume Tumor * *** *** 0 0

Day 17Day 20Day 23Day 26Day 29Day 32Day 35Day 38Day 41 Day 17Day 20Day 23Day 26Day 29Day 32Day 35Day 38Day 41 D. E. Nisch+/+ Nisch+/- Nisch+/+ Nisch+/- Cells Cells Cells Cells

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

F. *** *** G. +/- Tumor Stiffness Nisch+/+ Nisch 500 ** 20 Cells Cells 400 * 15 300 100µm

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Figure 7: Nischarin-Negative Cells Confer Resistance to Cell Cycle Control by Exosomes +/+ +/- +/- +/+ Nisch+/+ Cells w/ Nisch Cells Nisch+/- Cells Nisch Cells w/ Nisch Cells Nisch Cells B. +/- A. Nisch+/+ Exosomes w/ Nisch+/- Exosomes Nisch+/+ Exosomes w/ Nisch Exosomes

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

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20 C. *** No Exosomes +/+ D. E. 15 *** Nisch Exosomes Nisch+/- Exosomes

10 ***

5 Relative Absorbance Relative 0

Cells Cells +/+ +/- +/+ +/+ Nisch Nisch 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%)

Nisch+/- Cells Nisch+/- Cells +/+ +/- Nisch+/- Cells w/ Nisch w/ Nisch G. Exosomes Exosomes G1 (23.4%) G1 (85%) G1 (24.2%) S (34.7%) S (14.6%) S (33.7%) G2 (16.5%) G2 (21.9%) G2 (21%)

Exosomes from Nischarin-Expressing Cells Reduce Breast Cancer Cell Motility and tumor growth

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

Cancer Res Published OnlineFirst January 11, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-18-0842

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2019/01/11/0008-5472.CAN-18-0842.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

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