Cancer-associated fibroblasts produce matrix-bound vesicles that influence endothelial cell function

Intercellular communication between different cell types in solid tumors contributes to tumor growth and metastatic dissemination. The secretome of cancer-associated fibroblasts (CAFs) plays major roles in these processes. Using human mammary CAFs, we unveil a mechanism of cell-cell communication between CAFs with myofibroblast phenotype and endothelial cells (ECs) based on intercellular protein transfer through extracellular vesicles (EVs). CAFs transfer proteins to ECs, including plasma membrane receptors, which we have identified by using mass spectrometry- based proteomics. Using THY1 as an example of transferred plasma membrane-bound protein, we show that CAF-derived proteins can influence how ECs interact with other cell types. Here, we show that CAFs produce high amounts of matrix-bound EVs that have a key role in protein transfer. Hence, our work paves the way for further studies to understand how CAF-derived matrix-bound EVs influence tumor pathology by regulating functions of neighboring cancer, stromal and immune cells. One sentence summary CAFs with a myofibroblastic-like phenotype transfer proteins to ECs, including plasma membrane receptors, through matrix-bound EVs

molecule (Pecam1) to visualize ECs, showed RFP + endothelium in the lung metastases of these 121 mice ( Fig. 1G-H), but not in non-RFP expressing control mice (Fig. S1D). 122 Overall, our data provide evidences that CAFs communicate with ECs through the transfer of 123 proteins in vitro and possibly also in vivo.

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CAFs transfer plasma membrane receptors to ECs 125 To identify proteins that pCAFs transfer to HUVECs, we used a mass spectrometry (MS)-based 126 trans-stable-isotope labeling of amino acids in cell culture (trans-SILAC) proteomic approach (28). 127 First, we labeled the proteome of pCAFs with the heavy isotopologue of arginine and lysine, and 128 co-cultured them with unlabeled HUVECs for 4h or 24h. Then, we sorted the HUVECs and analyzed 129 their proteome by MS ( Fig. 2A). We quantified 808 and 1062 heavy-labeled proteins in at least 130 three out of five biological replicates at 4h and 24h time point, respectively ( Fig. 2B and Data S1).

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Of these, 698 proteins were common to both time points (Fig. 2B). Gene Ontology Cellular  Previous studies have shown that cancer and immune cells use EVs to transfer functional plasma 141 membrane proteins to ECs (38, 39). These types of proteins are highly relevant because they may 142 alter the function of the endothelium, including its interactions with surrounding cells. Therefore, 143 we focused our analysis on plasma membrane receptors and membrane-bound ligands. 144 Interestingly, the majority of the transferred membrane proteins were involved in immune protein in the HUVECs and referred to this value as "exogenous fraction". The exogenous fraction 150 ranges between 0 and 1, and the closer the value is to 1, the more the pCAF protein contributes 151 to the endothelial counterpart ( Fig. 2E and Data S1). CAF-derived Thy-1 membrane glycoprotein 152 (THY1) was the protein with the highest contribution to the HUVEC proteome, with an exogenous 153 fraction of 0.78 and 0.54 after 4h and 24h of co-culture, respectively ( Fig. 2E and Data S1). CD44 154 antigen (CD44) also contributed highly with an exogenous fraction of 0.46 and 0.35 at 4h and 24h, 155 respectively, and then integrin beta-3 (ITGB3), with an exogenous fraction of 0.21 at 24h of co-156 culture. The exogenous fraction for all the other receptors and ligands was lower than 0.15 (Fig. 157 2E and Data S1). Overall, these results indicate that mammary CAF-derived receptors/ligands can 158 quantitatively modify the proteome of the HUVECs. Next, we sought to determine whether 159 transferred proteins could functionally change the HUVECs and focused on THY1, since it 160 contributed to a major change of the HUVEC proteome.

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To confirm that the THY1 detected in HUVECs was derived from pCAFs, rather than being 163 expressed by HUVECs when co-cultured with them, we measured THY1 transcript in HUVECs in 164 monoculture and after 24h of co-culture with pCAFs. Using pCAFs as control of THY1 expressing 165 cells, we found that THY1 mRNA levels did not increase significantly in co-cultured HUVECs 166 compared to the monoculture (Fig. 3A). In addition, flow cytometry analysis confirmed the 167 transfer of THY1 from pCAFs to HUVECs (Fig. 3B-D). While THY1 was not present at the surface of 168 8 HUVECs in monoculture, after 24h of co-culture with pCAFs, the majority of HUVECs positively 169 stained for THY1 ( Fig. 3C-D). Moreover, pCAFs silenced for THY1 (Fig. 3B) transferred significantly 170 less receptor to the HUVECs (Fig. 3C-D), while the total amount of transferred proteins was not 171 affected (Fig. 3E).
172 THY1 (also known as CD90) is a glycophosphatidylinositol-anchored protein that localizes on the 173 extracellular side of the plasma membrane of cells and that binds to cancer cells and leukocytes 174 through plasma membrane receptors, such as integrin αvβ3, αvβ5, α5β1, αxβ2, αMβ2, syndecan-175 4, and adhesion G protein-coupled receptor (ADGRE5, also known as CD97) (45, 46). To assess the 176 function of pCAF-derived THY1, we measured leukocyte adhesion to HUVECs when co-cultured 177 with pCAFs silenced or not for THY1. Specifically, we used the human monocyte cell line THP-1 178 that expresses several THY1 binding partners ( Fig. 3F and Data S2). Microscopy analysis of the co-179 cultures showed that significantly fewer monocytes adhered to the HUVECs when co-cultured 180 with THY1-silenced pCAFs compared with control co-culture (siCtlr), supporting the functionality 181 of THY1 on the HUVEC surface (Fig. 3G). Hence, CAF-derived THY1 endows HUVECs with additional 182 cell-cell adhesion properties.

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MBVs have a major role in protein transfer to ECs 184 Next, we investigated how pCAFs transfer their proteins to HUVECs. Our data showed that a high 185 number of transferred proteins belonged to lipid bilayer-enclosed vesicles (Fig. 2C), supporting 186 that EVs can be a major route of intercellular protein transfer. It is known that CM-EVs are involved 187 in protein transfer (21, 22, 47-49), but the role of MBVs has not been investigated. 188 We isolated EVs from both the CM and extracellular matrix of pCAFs (Fig. 4A). Electron microscopy 189 analysis showed that the two EV types had a similar morphology (Fig. 4B). Nanoparticles tracking 190 analysis showed that the diameter of both types of EVs ranged between 50 and 350 nm (Fig. 4C).

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However, the amount and size distribution differed between the two EV types. Those in the CM 192 mainly included small particles with a diameter between 50 and 150 nm, while MBVs mostly 193 consisted of large EVs, with major peaks at 150 nm and 200 nm ( Fig. 4C-D). 194 We molecularly characterized pCAF-derived EVs using MS proteomics (Data S3). This analysis 195 confirmed that both types of particles contain common EV markers, such as tetraspanins CD63, 196 CD81, CD9 (30), and syntenin-1 (SDCBP) (50), but also highlighted differences, such as the relative 197 abundance of some EV markers and the presence of ADP-ribosylation factor 6 (ARF6) and tumor dataset, we selected proteins unique to each EV subpopulation and those with abundance 203 significantly different between the two. Then, we matched this subset to EV proteins whose 204 abundance was significantly different between CM-EVs and MBVs (Data S3). This analysis showed 205 that proteins typically found in large-medium EVs were generally more abundant in MBVs.

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Proteins typically found in small EVs, instead, were more abundant in CM-EVs (Fig. S2B). This 207 observation was consistent across the three datasets (Fig. S2B). Furthermore, proteins identified 208 only in CM-EVs displayed enrichment for endosome-related GOCC terms (Fig. S2C) that is one of 209 the documented intracellular origins of small EVs (Fig. S2A). Conversely, MBV unique proteins 210 displayed enrichment in GOCC terms associated with plasma membrane, cytosol, ER and 211 mitochondria (Fig. S2C), which are expected in large-medium EVs because of their biogenesis (30).

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Overall, these results further support that each extracellular compartment contains different 213 subsets of EVs.

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The majority of pCAF proteins transferred to ECs during co-culture was identified in both EV types 215 (Fig. 4F, Data S1 and S3), and their abundance positively correlated to the amount measured in 216 the EVs (Fig. 4G, Data S1 and S3). Notably, the majority of the transferred plasma membrane 217 receptors and membrane-bound ligands were more abundant overall in the MBVs ( Fig. 4H and   218 Data S3). 219 Next, we measured whether pCAF-derived CM-EVs and MBVs could transfer proteins to ECs. As 220 these EV types exist in different extracellular sites, we measured protein transfer when CAFs were 221 co-cultured in physical contact (direct co-culture) or not (indirect co-culture) with HUVECs ( Fig.   222 5A). In the direct co-culture, HUVECs are exposed to both types of EVs, while in the indirect co-223 culture to CM-EVs only (Fig. 5A). Strikingly, the amount of transferred proteins in the direct co-224 culture was more than two-fold higher compared with the indirect one (Fig. 5B). In line with this 225 result, HUVECs received significantly more proteins when treated with MBVs than with CM-EVs, 226 although the difference was less pronounced (Fig. 5C).

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Our MS analysis showed that THY1 was significantly more abundant in MBVs than in CM-EVs (Fig.   228 4H). We used direct and indirect co-cultures to measure THY1 transfer from pCAFs to HUVECs. As 229 for the total proteins, the transfer of THY1 mainly occurred when cells were in direct culture ( Fig.   230 5D). Moreover, upon treatment with MBVs, five-fold more HUVECs positively stained for THY1 231 compared with when treated with CM-EVs, and THY1 levels were two-fold higher (Fig. 5E).

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Together, our data provide evidences that MBVs are a major vehicle for protein transfer from 233 mammary CAFs to HUVECs. fibroblasts (21). Therefore, we compared protein transfer of our mammary CAFs with their 238 matched NFs isolated from the same patient (pNFs). pNFs are normal-like fibroblasts as they were 239 derived from macroscopically healthy tissue adjacent to the tumor. We found that pCAFs 240 transferred more proteins to HUVECs than pNFs (Fig. 6A), and confirmed this result using 241 microvascular endothelial cells (MVECs) (Fig. S3A). Since we showed that EVs are involved in 242 protein transfer, we compared the amounts of EVs released by pCAFs and pNFs. Nanoparticles

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Tracking Analysis showed that pCAFs deposited significantly more medium-large EVs in the ECM 244 than their NF counterpart (Fig. 4A, C-D). In the CM, instead, pNFs and pCAFs released EVs of similar 245 size and quantity. (Fig. 4C-D). Notably, HUVECs treated with CAF-derived MBVs received more 246 proteins than when treated with MBVs produced by pNFs (Fig. 6B) and CM-EVs and MBVs secreted 247 by pNFs transferred a comparable amount of proteins to HUVECs (Fig. 6C). Together, these results 248 suggest that CAFs transfer more proteins because they produce more MBVs. platelet-derived growth factor receptor alpha and beta (PDGFRA and PDGFRB), protein S100-A4 254 (S100A4, also known as FSP-1) and caveolin-1 (CAV1), with their protein transfer ability. 255 Intriguingly, we found that the amount of proteins transferred by fibroblasts significantly 256 correlated (P < 0.05) only with α-SMA protein levels (Fig. 6D). Microscopy analysis for α-SMA of 257 our pCAF lines confirmed the proteomic data showing that the pCAF1 line, which transferred more 258 proteins to ECs (Fig. S3B), contained more cells with high α-SMA protein levels than pCAF3 and 259 pCAF4 lines (Fig. 7A-B). To assess whether pCAFs expressing high or low protein levels of α-SMA 260 had different protein transfer ability, we needed to identify cell surface proteins to sort the two 261 living subpopulations for functional assays. To achieve this, we sorted CAFs with high and low α-262 SMA protein levels and analyzed them by MS proteomics (Fig. S4A). Principal component analysis 263 of 2,080 proteins quantified across the three pCAF lines separated the α-SMA low and α-SMA high 264 subpopulations ( Fig. 7C and Data S5). Moreover, 67 proteins showed difference in abundance 265 between α-SMA low and α-SMA high subpopulations in at least two of the three pCAF lines (Fig. S4B   266 and Data S5). Among those, there were 7 cell surface receptors (Fig. 7D). We followed up on the 267 tumor necrosis factor receptor superfamily member 12A (TNFRSF12A, also known as FN14,

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TweakR or CD266), because its levels were highly different between α-SMA low and α-SMA high CAFs 269 and it was a good candidate for cell sorting (Data S5). Immunofluorescence staining for α-SMA    subpopulations. To do this, we sorted TNFRSF12A high and TNFRSF12A low pCAFs, expanded them in 281 culture, and then assessed the expression of CAF markers by RT-qPCR. This analysis confirmed 282 that TNFRSF12A high pCAFs expressed higher levels of ACTA2 and other genes highly expressed in 283 mammary myCAFs (7, 16, 54), such as collagen alpha-1 (I) chain (COL1A1) and transgelin (TAGLN), 284 compared with TNFRSF12A low pCAFs ( Fig. S5A-B). Conversely, we did not detect significant 285 differences in mRNA levels of stromal cell-derived factor 1 (SDF1, also known as C-X-C motif 286 chemokine 12 or CXCL12) and interleukin-6 (IL6), which are highly expressed in mammary iCAFs 287 (7, 16, 54) ( Fig. S5A-B). Similarly, decorin (DCN), which has been found expressed at similar levels 288 13 in all CAFs (7, 54), had similar mRNA levels in our two sorted populations ( Fig. S5A-B). These data 289 suggest that high levels of the TNFRSF12A receptor are found in CAFs with myCAF phenotype.

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Consistent with this observation, in two publicly available single-cell RNA sequencing datasets of 291 CAFs isolated from patients with BC (7, 16), we found that both ACTA2 and TNFRSF12A mRNA 292 levels were high in the subpopulation defined by the authors as myCAFs (Fig. 8A-B). In addition,  . Notably, we found that CAFs transferred all these proteins to 326 ECs, and that CD44 and ITGB3 were among the receptors with the highest exogenous fraction.

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This suggests that, in addition to THY1, CD44 and ITGB3 may also influence EC functions.

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The ECM is an important source of signals that actively regulate tumor progression. Its structure  MBVs should be further investigated as a strategy to oppose cancer. For that, more work should 368 also be done to understand MBV biogenesis and function, a mechanism that so far has been 369 largely overlooked.    To measure the intercellular protein transfer in co-culture, donor cells were labeled with 10 μM  EVs were washed in PBS by two sequential steps of ultracentrifugation at 100,000 x g (4°C, 90min).

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The isolated EVs were used to treat 3x10 4 HUVECs in EGM-2. After 20h, HUVECs were detached To evaluate protein transfer by immunofluorescence, pCAFs were labeled with CellTrace TM CFSE 480 as described above and seeded on glass coverslips in a 24-well plate. Once they were adhered, 481 ECs were seeded in monoculture or in co-culture with pCAFs. After 24h, cells were fixed in 4% PFA.    For the proteome analysis of pCAFs and pNFs, cultured cells were washed with PBS, lysed in 2% 573 SDS in 100 mM Tris-HCl pH 7.4, incubated at 95°C for 5min, sonicated using a metal tip and 574 centrifuged at 16,000 x g for 10min. Lysates were mixed 1:1 with an internal standard composed 575 of a mix of SILAC heavy-labeled cCAFs/cNFs. Protein lysate was mixed with NuPAGE TM LDS sample 576 Buffer (4x) and 1 mM DTT. Proteins were separated using 4-12% gradient NuPAGE TM Novex Bis-577 Tris gel, which then was stained with Coomassie Blue. Gel lanes were cut into slices, proteins were 578 in gel digested with trypsin (76), peptides were desalted using C18 StageTip and analyzed by MS.

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The emitter was kept at 50°C (trans-SILAC) or 35°C (pCAF1 proteome) by means of a column oven 588 integrated into the nanoelectrospray ion source (Sonation).

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Eluting peptides were electrosprayed into the mass spectrometer using a nanoelectrospray ion 590 source (Thermo Fisher Scientific). An Active Background Ion Reduction Device (ABIRD, ESI source 591 solutions) was used to decrease air contaminants signal level.   The proteomic data of EVs were downloaded from three publicly available datasets (51-53). For 666 each dataset, we considered the proteins unique to each EV subpopulation and those with 667 abundance significantly different between the two as statistically analyzed by the authors, except 668 that for (53), for this paper we considered proteins with at least a two-fold change and P < 0.05.

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The selected proteins were matched by gene name with the proteins whose abundance was 670 significantly different between CM-EVs and MBVs (two-sided Student's T-test, P < 0.05). The Z-671 score was calculated by row.   Mice were culled three weeks after the injection. A small incision was made in the trachea and 1 693 ml of 2% low-melting point agarose was introduced slowly into the lungs through a 22G needle.

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Lungs were excised and fixed in 4% PFA for 2h at 4°C. Then, lungs were sliced into 300 μm thick  Samples were left to absorb onto carbon surfaces for 30min. Grids were floated on 100 μl droplets 713 of PBS followed by fixation on a 50 μl droplet of 1% Glutaraldehyde (Agar Scientific Ltd) for 5min.

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Grids were washed with distilled water before contrast staining with Uranyl Oxalate (Merck UK) 715 pH 7.0 (10min in the dark) followed by Methylcellulose/Uranyl Acetate (Merck UK) embedding 716 (10min on ice in the dark). Grids were scooped up on Platinum Loops and excess fluid gently 717 31 drained off leaving thin films. Grids were then left to dry before picking off and storing in a grid 718 box. Samples were viewed on a JEOL 1200 EX TEM running at 80 kV and digital images were 719 captured using Olympus ITEM software and a Cantega 2kx2k Camera.

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Staining and confocal microscopy of human mammary tumors 721 Human mammary tumors were obtained through NHS Greater Glasgow and Clyde Bio-repository.

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Formalin-fixed paraffin-embedded tissues were cut into 4 μm thick slices. Nine independent 723 patient samples underwent high-pH antigen retrieval prior to immunofluorescence staining.