IFIT3 (interferon induced protein with tetratricopeptide repeats 3) modulates STAT1 expression in small extracellular vesicles

We have previously shown that the αvβ6 integrin plays a key role in promoting prostate cancer (PrCa) and it can be transferred to recipient cells via small extracellular vesicles (sEVs). Furthermore, we have reported in a proteomic analysis that αvβ6 integrin down-regulation increases the expression of IFIT3 (interferon induced protein with tetratricopeptide repeats 3) in PrCa cells and their derived sEVs. IFIT3 is a protein well known for being an antiviral effector, but recently its role in cancer has also been elucidated. To study the relationship between IFIT3 and STAT1 (signal transducer and activator of transcription 1), an upstream regulator of IFIT3, in PrCa cells and their released sEVs, we used CRISPR/Cas9 techniques to down-regulate the expression of the β6 integrin subunit, IFIT3 or STAT1. Our results show that IFIT3 and STAT1 are highly expressed in PrCa cells devoid of the β6 integrin subunit. However, IFIT3 but not STAT1, is present in sEVs derived from PrCa cells lacking the β6 integrin subunit. We demonstrate that loss of IFIT3 generates sEVs enriched in STAT1 but reduces the levels of STAT1 in the cells. As expected, IFIT3 is not detectable in STAT1 negative cells or sEVs. We thus propose that the observed STAT1 enrichment in sEVs is a compensatory mechanism for the loss of IFIT3. Overall, these results provide new insights into the intrinsic role of IFIT3 as a regulator of STAT1 expression in sEVs and in intercellular communication in PrCa.


Introduction
5'GCGCTGGCGGGCGTTGGCGT) (Synthego Corporation, Redwood City, CA). Each gRNA was complexed with Cas9 (Synthego Corporation, Redwood City, CA) in TE buffer for 15 minutes, with a gRNA: Cas9 ratio of 250:50 pmols. Complexed ribonucleoprotein particles (RNPs) were transfected in PC3-WT cells using a nucleofector 4d X-unit following the manufacturer's guidelines for a 100 μL reaction (Lonza, Basel, Switzerland). After 3 days of recovery, the cells were single-cell sorted using a FACS Aria 2 (BD, Franklin Lakes, NJ) into 96-well plates and allowed to expand for several weeks. Genomic DNA from each clone was isolated, purified, and PCR amplified across the CRISPR target sites and Sanger sequenced. Sanger sequencing deconvolution was carried out using DECODR (DECODR.org) to assess the indel spectrum of each clone. STAT1 genomic depletion resulted in PC3-STAT1KO CRISPR clones referred to as STAT1KO C21 and STAT1KO C1C5. IFIT3 genomic depletion resulted in PC3-IFIT3 CRISPR clones referred to as IFIT3KO C13 and IFIT3KO C15. Finally, for the β3 integrin subunit genomic depletion, one of the clones was unsuccessful, and therefore used as our CRISPR control cells, referred to as PC3-CRISPR control.

Immunoblot analysis
For immunoblot analysis (IB), the total cell lysates (TCL) and sEV lysates were prepared using radio immuno-precipitation assay (RIPA) buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100 and 1% sodium deoxycholate) supplemented with protease inhibitors such as calpain, aprotinin, leupeptin, pepstatin, and sodium fluoride. Protein concentration was determined using the BioRad DC TM protein assay kit according to the manufacturer's protocol. Equal amounts of TCL or sEV lysates under reducing (heated with 2-mercaptoethanol) or nonreducing (heated without 2-mercaptoethanol) conditions were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane (immobilon-E PVDF membrane, pore size 0.45 µm, Millipore). The membrane was incubated with blocking buffer (5% non-fat dry milk in Tris Buffer Saline with 0.1% Tween 20 [TBS-T]) for 1 hour at room temperature. The membrane was incubated overnight with respective primary antibodies (Abs) followed by three TBS-T washes of 5 minutes each at room temperature.
To visualize the protein, the membrane was incubated with WesternBright TM ECL (horseradish peroxidase) HRP substrate kits (Advansta Inc., CA, USA).

Small extracellular vesicle isolation
Iodixanol density gradients isolation sEVs isolated by differential ultracentrifugation (described above) from PC3-WT, PC3-CRISPR control, β6KO C3, β6KO C5, β6KO C7, IFIT3KO C13, IFIT3KO C15, STAT1KO C21 and STAT1KO C1C5 cells, were further isolated using iodixanol density gradient ultracentrifugation as previously described [44]. Briefly, a 60% stock solution of iodixanol (OptiPrep™, Sigma # 1556) was mixed in a 1:1 ratio with a buffer (0.25 M sucrose, 10 mM Tris pH 8.0, and 1 mM EDTA, pH 7.4) to obtain a 30% iodixanol solution. sEVs were mixed with the 30% iodixanol solution and layered at the bottom of an ultracentrifuge SETON tube. Next, 700 μL of 20% iodixanol and 700 μL of 10% iodixanol solutions were layered on top of the 30% iodixanol-sEV suspension to create a discontinuous gradient. Samples were centrifuged for 70 minutes at 350,000 x g, 4°C in a SW55Ti rotor using a Beckman L8-70 M ultracentrifuge. A total of 10 consecutive fractions of 260 μL each were collected from top to bottom of the gradient. The refractive index of each fraction was assessed using an ABBE-3 L refractometer, (Fisher Scientific) and their density was calculated. All 10 fractions were diluted with 1 mL of PBS and centrifuged for 70 minutes at 100,000 x g, 4°C in a TLA-100.2 rotor using a Beckman, Optima TL ultracentrifuge. The pellets obtained from each of the 10 fractions were washed in 1 mL of PBS, as specified above. The final pellet of all fractions was resuspended in 65 μL of PBS and stored at −80°C.

Nanoparticle tracking analysis (NTA)
NTA was used to determine the size distribution and concentration of sEVs derived from PC3-WT, PC3-WT cells treated with NS or D2 siRNAs as well as PC3-CRISPR control, β6KO C3, β6KO C5, β6KO C7, IFIT3KO C13, IFIT3KO C15, STAT1KO C21 and STAT1KO C1C5 cells. The samples were loaded in the instrument manually, and the Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20210580/921484/bcj-2021-0580.pdf by guest on 10 October 2021 analysis was performed according to the manufacturer's instructions using the NTA software (NS300, Malvern Instruments). The temperature for all experiments was 25°C. sEV suspensions of differential ultracentrifugation-isolated sEVs and iodixanol density gradient-isolated sEV fractions were diluted in PBS at a ratio of 1:1000 and/or 1:200, respectively. The pooled sEV fractions (2)(3)(4)(5) were combined and then diluted 1:200 in PBS for NTA analysis. The analysis of size distribution and concentration of sEVs was performed under a detection threshold of 5. Video files were captured for a duration of 30 seconds (repeated 3 times) with a frame rate of 30 frames per second using the NS300 software version 3.1.54.

Transmission electron microscopy (TEM)
The pooled iodixanol density gradient-isolated sEV fractions (2-5) derived from PC3-CRISPR control and β6KO C5 cells were combined and then diluted 1:200 in PBS for TEM analysis as previously described [17]. Briefly, a 5 µL volume of sEVs suspended in 10 mM TRIS-EDTA solution (pH 7.8) was applied to a thin carbon grid that was glow discharged for 2 minutes using a Pelco Easyglow instrument. A 5 µL volume of freshly made 2% uranyl acetate stain solution was applied and incubated for 2 minutes on the grid. Excess sample and stain were removed using a Whatman filter paper. The staining process was repeated, and the grid was allowed to dry until imaged. TEM micrographs were collected using a Tecnai T12 TdEM microscope at 100KeV. The images were recorded on a Gatan Oneview 4Kx4K camera. Each image was collected by exposing the sample for 4 seconds. A total of 100 dose fractionated images were collected into a single micrograph. The imaging data were collected at 1.5 to 2 microns under focus.

Statistical Analysis
The means and standard deviations of size and concentration distributions of sEV preparations measured using NTA derived from the β6 integrin subunit-expressing PC3-WT, PC3-CRISPR control, β6KO C7, β6KO C3, IFIT3KO C13 or IFIT3KO C15 cells were analyzed. Student's t-test was used for comparing two group means. A twosided P value of ≤ 0.05 is considered statistically significant. Software GraphPad Prism 7 was used for data analysis.

IFIT3 and STAT1 protein expression is increased upon downregulation of the β6 integrin subunit in PrCa cells
We have previously shown that downregulation of the αvβ6 integrin in PrCa cells results in increased protein expression of STAT1 in total cell lysates (TCL) [24]. We now show that downregulation of the β6 integrin subunit in PrCa cells also results in elevated IFIT3 protein expression in TCL ( Figure 1). We downregulated the β6 integrin subunit in PC3 wild-type (PC3-WT) cells using a siRNA that specifically targets the β6 integrin subunit ( Figure 1A). PC3-WT cells were treated with either non-silencing siRNA (NS) or the β6 integrin subunit siRNA (D2) ( Figure 1A, left panel). IFIT3 and STAT1 levels in TCL for each siRNA treatment condition were determined by immunoblotting (IB) analysis ( Figure 1A, right panel). We find that compared with the NS siRNA treatment, the levels of IFIT3 and STAT1 are upregulated upon treatment with the D2 siRNA. TSG101 and Actin were included as loading controls, respectively ( Figure 1A, left and right panels). In parallel, to assess the protein levels of IFIT3 and STAT1 in PrCa cells, we used PC3-WT and PC3 cells harboring CRISPR/Cas9-mediated downregulation of the β6 integrin subunit. CRISPR/Cas9 treatment generated different PC3-β6KO CRISPR clones referred to as β6KO C5 and β6KO C7 ( Figure 1B, left panel). As expected, IB analysis of TCL from β6KO C5, β6KO C7 and β6KO C3 cells ( Figure 1B, left panel, and data not shown) results in increased protein expression of IFIT3 and STAT1 compared to PC3-WT cells ( Figure 1B, right panel). Actin was included as a loading control ( Figure 1B, left and right panels).

IFIT3 is detected in sEVs isolated from PrCa cells devoid of the β6 integrin subunit
Proteomic analysis performed by our group has previously demonstrated that αvβ6 integrin negatively regulates STAT1 and IFIT3 protein levels in both PC3 PrCa cells and their sEVs [24].
We first assessed the protein levels of IFIT3 in sEVs derived from the β6 integrin subunit expressing PC3-WT cells and from β6KO C7 cells that lack the β6 integrin subunit ( Figure 2) by using IB analysis. As expected from previous studies [17], we find that the β6 integrin subunit is expressed in PC3-WT sEV fractions 1 to 5 which correspond to a density range of 1.103-1.169 g/mL ( Figure 2A, left panel). The tetraspanins, CD63 and CD81, which are known sEV markers [19], are also expressed in the same fractions containing the β6 integrin subunit (Figure 2A, left panel). Not surprisingly, we find that IFIT3 and STAT1 protein expression is absent in PC3-WT sEV fractions ( Figure 2A, right panel). Moreover, the tetraspanin CD9, a canonical sEV marker [19], is expressed in fractions 1 to 3, which collectively have a density range of 1.103-1.134 g/mL ( Figure 2A, right panel). The endoplasmic reticulum marker (ER), calnexin (CNX) [45], is absent in all the sEV fractions, but present in PC3-WT and β6KO C7 TCL (Figure 2A, right panel).
CNX absence excludes the possibility of contamination of ER proteins in the isolated sEVs [19]. These data demonstrate that the presence of the β6 integrin subunit in PrCa cells, prevents IFIT3 expression or localization in PrCa-derived sEVs.
In the sEV fractions derived from β6KO C7 cells, we confirm the absence of the β6 integrin subunit ( Figure 2B, left panel). Meanwhile, the sEV markers CD63 as well as CD81 are found to be expressed in sEV fractions 2 to 5 which cover a density range of 1.099-1.151 g/mL ( Figure 2B, left panel). In the absence of the β6 integrin subunit, IFIT3 is expressed in sEV fractions 2 to 4, which is also a reproducible finding in β6KO C3 cells ( Figure 2B, right panel, and data not shown). However, STAT1 expression is not detected in any of the sEV fractions derived from β6KO C7 cells ( Figure 2B, right panel). We also find that CD9 is expressed in sEV fractions 2 to 5 ( Figure 2B, right panel). The absence of CNX in the sEV fractions confirms the lack of ER proteins in the isolated sEVs [19] ( Figure 2B, right panel). These results further establish that the αvβ6 integrin plays a role in downregulating IFIT3 protein expression in PrCa cell-derived sEVs.
Next, we assessed the size and concentration of sEV fractions using nanoparticle tracking analysis (NTA). We isolated sEVs from PC3-WT or β6KO C7 cells by differential ultracentrifugation (100,000 x g) [44], which are referred to as PC3-WT Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20210580/921484/bcj-2021-0580.pdf by guest on 10 October 2021 100K or β6KO C7 100K. Iodixanol density gradients were used to isolate sEV fractions from PC3-WT 100K or β6KO C7 100K sEVs and remove contaminants by separating the sEVs on different buoyant densities. We collected 10 sEV fractions from top to bottom of the iodixanol density gradient. We then proceeded to assess the size and concentration of sEV fractions derived from PC3-WT or β6KO C7 cells (Figure 3). The mean sizes of sEV fractions 2 to 5 derived from PC3-WT are 205.6 nm, 209.4 nm, 211.4 nm, and 207.5 nm, respectively ( Figure 3A). The mean sizes of sEV fractions 2 to 5 derived from β6KO C7 cells are 185.1 nm, 191.2 nm, 204.1 nm and 167.2 nm, respectively ( Figure 3B). These results align with our previous research findings where we determined that sEVs derived from PrCa cells are less than 200 nm in size [16,46]. Furthermore, we did not detect significant concentration changes in sEV preparations derived from the β6 integrin subunit-expressing PC3-WT cells (n=2) compared to sEV fractions derived from the PC3-CRISPR clones devoid of the β6 integrin subunit, β6KO C7 (n=2) or β6KO C3 (n=2, data not shown). Specifically, the mean concentration of two sEV preparations that comprise fractions 2-5 derived from PC3-WT, β6KO C7 or β6KO C3 is 6.51 x 10 8 , 1.28 x 10 9 , or 7.99 x 10 8 particles/mL, respectively ( Figure 3A-B). Similar results were obtained for sEVs isolated via differential ultracentrifugation (100,000 x g) from the PC3-CRISPR control cells expressing the β6 integrin subunit, or PC3-β6KO CRISPR clones (Supplementary Figure S1).

Downregulation of the β6 integrin subunit in PrCa cells does not affect sEV size distribution
It has been established that sEVs exhibit a size range of 50-200 nm [47]. Therefore, we wanted to assess whether the absence of the β6 integrin subunit had any impact on sEV size. In order to answer this question, we assessed the sEV size by NTA. Using the PC3-CRISPR control cells that express the β6 integrin subunit or the PC3-CRISPR clones devoid of the β6 integrin subunit β6KO C5 and β6KO C7, we isolated sEVs via density gradients and pooled sEV fractions 2 to 5. Noteworthy, the β6 integrin subunit positive cells such as the PC3-WT cells, as well as PC3-CRISPR control cells that endogenously express the β6 integrin subunit, depict comparably low levels of IFIT3 and STAT1. NTA analysis shows that the mean sizes of pooled sEV fractions 2 to 5 derived from the PC3-CRISPR control, β6KO C5, and β6KO C7 cells are 188.8 nm, 165.5 nm, and 193.3 nm, respectively ( Figure 4A). Transmission electron microscopy (TEM) analysis displays the size and morphology of density gradient-isolated sEVs derived from the PC3-CRISPR control and β6KO C5 cells ( Figure 4B). According to the TEM analysis of sEV fractions derived from PC3-CRISPR control cells, 76% of the sEVs are < 100 nm and 24% are between 100 and 200 nm in size. Specifically, out of 41 sEVs derived from PC3-CRISPR control cells, 31 had a size < 100 nm and 10 had a size between 100 nm to 200 nm. For sEV fractions derived from β6KO C5 cells, 82% of the sEVs are < 100 nm and 18% are between 100 nm to 200 nm in size. Specifically, for sEVs derived from β6KO C5 cells, we counted a total of 55 sEVs in which 45 are < 100 nm in size and 10 are between 100 to 200 nm in size. As a result, we concluded that there is no significant size difference between sEVs containing or devoid of the β6 integrin subunit. It has been previously shown that STAT1 regulates IFIT3 expression, as STAT1 is a key protein needed for relaying the transcriptional activity of the ISG proteins [38]. However, whether IFIT3 has any regulatory effect on STAT1 expression is not understood. To analyze this potential interaction, we utilized PrCa cells harboring CRISPR/Cas9-mediated downregulation of IFIT3 (IFIT3KO clone C13 and IFIT3KO clone C15) as well as PC3-CRISPR control cells. First, we isolated sEVs from IFIT3KO C13 and PC3-CRISPR control cells via differential ultracentrifugation, designated as IFIT3KO C13 100K and PC3-CRISPR control 100K sEVs. After this step, we further isolated IFIT3KO C13 100K and PC3-CRISPR control 100K sEVs using density gradients and characterized the sEV size distribution by NTA ( Figure 5). We find that the mean sizes of individual sEV fractions 2 to 5 derived from PC3-CRISPR control cells are 197.8 nm, 189.7 nm, 212.7 nm, and 208.0 nm, respectively ( Figure 5A). Furthermore, IFIT3KO C13 sEV fractions 2 to 5 have mean sizes of 201.1 nm, 251.1 nm, 201.5 nm, and 219.6 nm, respectively ( Figure 5B). These results show that the sEVs isolated from PC3-CRISPR control or IFIT3KO C13 cells fall within the reported sEV size range [47] and are not affected by CRISPR-based gene editing.
We then proceeded to analyze the content of sEVs derived from the PC3-CRISPR control cells and the PC3-CRISPR clones devoid of IFIT3 (IFIT3KO C13 and IFIT3KO C15). IB analysis of IFIT3KO C13 and IFIT3KO C15 TCL confirms the absence of IFIT3 ( Figure 6A). Moreover, STAT1 expression is decreased in IFIT3KO C13, and IFIT3KO C15 cells compared to PC3-WT cells ( Figure 6A). Actin was used as a loading control ( Figure 6A). PC3-CRISPR control cell-derived sEV fractions 1 to 4, which have a density range of 1.099 -1.137 g/mL, contain the β6 integrin subunit as well as the sEV markers CD63 and CD81 ( Figure 6B, left panel). Furthermore, IFIT3 as well as STAT1 expression is absent in all sEV fractions ( Figure 6B, right panel). The sEV markers TSG101 and CD9 are specifically detected in fractions 2 to 5 corresponding to a density range of 1.113-1.148 g/mL ( Figure 6B, right panel). CNX absence in all 10 sEV fractions confirms the lack of contamination of ER proteins in our isolated sEVs ( Figure 6B, right panel).
In sEV fractions derived from the PC3-CRISPR clone devoid of IFIT3, termed IFIT3KO C13, the β6 integrin subunit as well as CD63 and CD81 are abundant in fractions 1 to 3, which span the density range of 1.106-1.130 g/mL ( Figure 6C, left panel). CNX is absent in all 10 sEV fractions, but present in IFIT3KO C13 TCL ( Figure  6C, left panel). Furthermore, STAT1 expression is clearly observed in sEV fractions 2 to 4 derived from IFIT3KO C13 and IFIT3KO C15 cells ( Figure 6C, left panel and data not shown), which cover a density range of 1.110-1.134 g/mL ( Figure 6C, right panel). The levels of TSG101 and CD9 are enriched in the sEV fractions compared to IFIT3KO C13 TCL ( Figure 6C, right panel). These results suggest that there is a positive feedback loop interaction in which STAT1 compensates for the loss of IFIT3. Therefore, our results show that STAT1 packaging is increased in sEVs derived from PrCa cells devoid of IFIT3.

STAT1 downregulation affects IFIT3 expression in PrCa cells and their sEVs
Our results prompted us to assess IFIT3 protein expression levels in PrCa-derived sEVs in the absence of STAT1.
For this purpose, we used PrCa cells harboring CRISPR/Cas9-mediated downregulation of STAT1, thus generating STAT1KO C21 and STAT1KO C1C5 CRISPR clones. We first proceeded to isolate sEVs from STAT1KO C21 cells via differential ultracentrifugation (STAT1KO C21 100K). Then, we isolated sEVs from STAT1KO C21 cells using density gradients. IB analysis of TCL from PC3-CRISPR control cells and the PC3-CRISPR clones devoid of STAT1, STAT1KO C21 and STAT1KO C1C5, confirms the lack of STAT1 expression in STAT1KO clones compared to PC3-CRISPR control cells ( Figure 7A). Noteworthy, IFIT3 expression is absent in both STAT1KO C21 and STAT1KO C1C5 cells ( Figure 7A). These results are consistent with previous studies that posit STAT1 as a key player in regulating the expression of the ISG proteins such as IFIT3 [37]. Using NTA analysis, we characterized the size distribution of density gradient-isolated sEVs derived from STAT1KO C21 cells ( Figure 7B). The mean sizes of sEV fractions 2 to 5 derived from STAT1KO C21 cells are 168.3 nm, 152.8 nm, 170.9 nm, and 154.5 nm, respectively. Furthermore, we did not detect significant concentration changes in sEV preparations derived from the PC3-CRISPR control cells ( Figure 5A) when compared to sEV preparations derived from the PC3-CRISPR clones devoid of STAT1, STAT1KO C21 or STAT1KO C1C5 ( Figure 7B). Thus, we show that IFIT3 downregulation does not affect sEV release.
We then proceeded to analyze STAT1KO C21 derived sEV fractions by IB. According to the IB analysis, STAT1 and IFIT3 are absent in all sEV fractions derived from the STAT1KO C21 and STAT1KO C1C5 cells ( Figure 7C and, data not shown). The sEV markers TSG101, CD9 and CD81 are abundant in sEV fractions 2 to 4, which collectively represent a density range of 1.123-1.144 g/mL. CNX is absent in all 10 sEV fractions, but expressed in β6KO C7 and STAT1KO C21 TCL. These data establish that knocking down STAT1 in PrCa cells results in IFIT3 downregulation in STAT1KO cell-derived sEV fractions. Our results further show that STAT1 downregulation causes the ablation of IFIT3 expression in PrCa cells and their sEVs.

Discussion
Our study reveals that downregulation of the β6 integrin subunit via siRNA (D2) or CRISPR/Cas9 techniques in PrCa cells results in increased protein expression of IFIT3 and STAT1 in PrCa cells. IFIT3 expression, but not STAT1, is increased in density gradient-isolated sEVs derived from PrCa cells devoid of the β6 integrin subunit. We demonstrate that loss of IFIT3 generates sEVs enriched in STAT1, but reduces the levels of STAT1 in the cells. As expected, IFIT3 is not detectable in STAT1 negative cells or sEVs. Overall, these results demonstrate for the first time that IFIT3 plays a novel role in regulating STAT1 expression in PrCa cells and their sEVs. We propose that STAT1 enrichment in PrCa-derived sEVs devoid of IFIT3 is a compensatory mechanism for IFIT3 loss. Previously, our group has shown the relevance of the αvβ6 integrin in cancer progression by establishing that this integrin can be transferred from PrCa cells to recipient cells via sEVs [16,17]. We have also demonstrated that the αvβ6 integrin contained in PrCa cell-derived sEVs plays an unequivocal role in promoting cell proliferation, survival, and angiogenesis [16,25,26]. Moreover, proteomic analysis previously published by our group, shows that the αvβ6 integrin plays a role in downregulating the protein expression of different ISG such as IFIT3 in PrCa cells as well as in the sEVs released by these cells [24]. Results from our current study align with these findings by showing that IFIT3 is expressed in sEVs derived from PrCa cells devoid of the β6 integrin subunit whereas IFIT3 is absent in sEVs derived from PrCa cells that contain the β6 integrin subunit.
Aside from its antiviral activity, the role of IFIT3 in cancer is poorly understood, and even less so in PrCa. However, a study conducted by Weichselbaum and coworkers showed that IFIT3 was downregulated in PrCa human tumors [48]. Moreover, IFIT3 expression has been shown to be detrimental for patient outcomes in other cancers. In oral squamous cell carcinoma (OSCC), for instance, IFIT3 ectopic expression has been shown to correlate with poor survival in OSCC patients [39]. In pancreatic ductal carcinoma (PDAC), IFIT3 expression was proposed to be a prognostic marker of PDAC, as well as shown to mediate antiapoptotic activity and chemoresistance of PDAC cells [49]. Notwithstanding, our findings are concomitant with other research supporting an anti-proliferative role of IFIT3 in cancer [42], as we demonstrate a new role for the αvβ6 integrin as a negative regulator of IFIT3. For example, in pro-monocytic myeloid leukemia cells, IFIT3 expression induced G1 phase arrest by increasing the expression of the cyclin-dependent kinase inhibitors, p21 and p27 [37]. In another study, it was shown that IFIT3 overexpression in lung cancer cells was able to exert an anti-tumor activity [50]. In fact, IFIT3 overexpression caused the downregulation of NFκB pathways, cyclin D1, c-Myc, and PCNA. IFIT3 has also been shown to reduce lung cancer cell proliferation, migration, and epithelial mesenchymal transition [42]. The same study demonstrated that IFIT3 tumor-suppressor function was dependent on increased expression of p53. Moreover recently, Wang et al., showed that the expression levels of IFIT3 in the peripheral blood from patients with acute promyelocytic leukemia is low compared to healthy controls [51]. Therefore, we hypothesize that αvβ6 integrin downregulates IFIT3 as a mechanism to promote tumor growth. Thus, in this study, we elucidate this newfound role of the αvβ6 integrin as a negative regulator of IFIT3.
We also show for the first time that STAT1 is detected in sEVs derived from PrCa cells devoid of IFIT3 and isolated via density gradients. Noteworthy, Cossetti et al., detected STAT1 in density gradient-isolated sEVs from neural stem/precursor cells only by prior cell-stimulation with pro-inflammatory cytokine cocktails [52]. In our results, however, we observe STAT1 expression in sEVs without exposing our cells to proinflammatory cytokines and more importantly, in the absence of IFIT3. Abundant STAT1 expression in PrCa-derived sEVs devoid of IFIT3 in the absence of cytokine stimulation, might be a cell line-specific effect, since neural stem/precursor cells might require higher levels of stimulation to induce IFN proteins compared to PrCa cells. Overall, these results suggest that there is a compensatory mechanism in which increased expression of STAT1 counterbalances the loss of IFIT3. Moreover, STAT1 is known to have a tumor suppressor role [31][32][33] whereas IFIT3 has been shown to play a dual role in cancer [39,42]. Therefore, we speculate that the relationship between STAT1 and IFIT3 in this study may suggest a mechanism of balance between proliferative and anti-proliferative role of IFIT3 in a context-dependent manner.
In the literature, it is well known that STAT1 induces the expression of IFIT proteins by serving as a component of the heterotrimeric ISGF3 transcription complex [38]. Other research has shown that IFIT3 associates with STAT1 and STAT2 to mediate their interaction, heterodimerization, and enhance STAT1 and STAT2 nuclear translocation [41]. However, no other studies have presented evidence of IFIT3 negative regulation of STAT1 expression and increased loading into sEVs.
The mechanism by which IFIT3 regulates STAT1 in PrCa needs to be further explored. For our proposed model (Figure 8), we speculate that exacerbated expression of STAT1 in sEVs mediated by IFIT3 downregulation is potentially occurring through any of the following mechanisms: (1) transcriptional, (2) translational, or (3) post-translational levels. As a consequence of these modifications, (4) increased STAT1 loading into multivesicular bodies (MVBs) results in STAT1 enrichment in sEVs released by PrCa cells.
As sEVs play a pivotal role in cell-cell communication and have the potential to alter recipient cells' phenotype and function [23][24][25], we therefore believe that PrCaderived sEVs containing the proteins STAT1 or IFIT3, have an important physiological relevance. STAT1 for instance, is known to mediate macrophage M1 polarization [53,54].
Along the same lines, IFIT3 has been described as a novel marker of M1 macrophages [55]. Therefore, we foresee that transfer of sEVs containing IFIT3 or STAT1 to other cells in the TME, such as macrophages, might change their surface receptor expression as well as their cytokine release into a M1 pro-inflammatory response.
In summary, our results demonstrate that loss of IFIT3 generates sEVs enriched in STAT1. We thus propose that the observed STAT1 enrichment in sEVs is a compensatory mechanism for the loss of IFIT3. These results open new promising avenues to explore the intrinsic role of PrCa-derived sEVs containing IFIT3 or STAT1, and how transfer of these cargoes to neighboring non-cancerous cells affects their phenotype as well as their function.

Data Availability
All of the primary data that is presented in this study can be requested in electronic form by contacting Dr. Lucia Languino (lucia.languino@jefferson.edu).

Disclosure Statement
The authors declare no conflict of interest.

Funding
This study was supported by NCI P01-CA140043, R01-CA224769, to LRL. CRISPR/Cas9-mediated downregulation of β6 integrin subunit, STAT1, and IFIT3. We thank Sudheer Molugu from the Electron Microscopy Resource laboratory at the University of Pennsylvania for generating the TEM figures. We would also like to thank Dr. Paul Weinreb and Dr. Sheila Violette for developing the antibody against the β6 integrin subunit (6.2A1). We are thankful to Shiv Ram Krishn, Fabio Quaglia, Christopher Shields and Vaughn Garcia for helping with the preparation of this manuscript. We are thankful to Dr. Benovic's laboratory for sharing equipment with us and to Sophia Green, Mark Fortini, and Jennifer Wilson for help with the preparation of the manuscript. We are also thankful with BioRender.com for providing us with tools to generate Figure 8.