Analysis of sialyl-Lewis x on MUC5AC and MUC1 mucins in pancreatic cancer tissues

Analysis of sialyl-Lewis x on MUC5AC and MUC1 mucins in pancreatic cancer tissues. Meritxell Balmaña1,2, Adrià Duran1, Catarina Gomes2, Esther Llop1, Raquel López-Martos5, M. Rosa Ortiz5, Sílvia Barrabés1, Celso A. Reis2,3,4#, Rosa Peracaula1# 1Biochemistry and Molecular Biology Unit, Department of Biology, University of Girona, Girona, Spain. 2Instituto de Investigação e Inovação em Saúde, I3S; and Institute of Molecular Pathology and Immunology of University of Porto, Ipatimup; Porto, Portugal. 3Medical Faculty, University of Porto, Al. Prof. Hernâni Monteiro, 4200 – 319 Porto, Portugal 4Instituto de Ciências Biomédicas de Abel Salazar - ICBAS, University of Porto, Porto, Portugal 5Department of Anatomic Pathology, Dr. Trueta University Hospital, Girona, Spain. #Correspondence should be addressed to Celso A. Reis, IPATIMUP/i3S, Rua Alfredo Allen, 208 4200-135 Porto - Portugal, E-mail: [email protected] . Tel: +351 220408800; Fax: 351 225570799 or Rosa Peracaula, Biochemistry and Molecular Biology Unit, Department of Biology, University of Girona, 17071 Girona, Spain. E-mail: [email protected]. Tel:+34 972418370; Fax: +34 972418150. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 Abstract Pancreatic adenocarcinoma (PDAC) lacks efficient biomarkers. Mucins are glycoproteins that can carry aberrant glycosylation in cancer. Our objective was to identify cancer-related glycan epitopes on MUC1 and MUC5AC mucins in PDAC as potential biomarkers. We have analysed the tumourassociated carbohydrate antigens sialyl-Lewis x (SLex) and sialyl-Tn (STn) on MUC1 and MUC5AC in PDAC tissues. The selected cohort for this study consisted of twenty-one PDAC tissues positive for SLex antigen and three normal pancreas specimens as controls. STn expression was shown in 76% of the PDAC tissues. MUC1 and MUC5AC were detected in 90% of PDAC tissues. We performed in situ proximity ligation assay combining antibodies against mucins and glycan epitopes to identify specific mucin glycoforms. MUC1-SLex and MUC5AC-SLex were found in 68% and 84% respectively, of the mucin expressing PDAC tissues, while STn hardly colocalized with any of the evaluated mucins. Further analysis by Western blot of MUC5AC and SLex in eight PDAC tissue lysates showed that six out of eight cases were positive for both markers. Moreover, immunoprecipitation of MUC5AC from positive PDAC tissues and subsequent SLex immunodetection confirmed the presence of SLex on MUC5AC. Altogether, MUC5AC-SLex glycoform is present in PDAC and can be regarded as potential biomarker. Keywords: mucins; pancreatic cancer; sialyl-Lewis x. Abbreviations: PDAC, pancreatic cancer; PLA, in situ proximity ligation assay; SLex, sialyl-Lewis x; STn, sialyl-Tn 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 1. Introduction Pancreatic adenocarcinoma (PDAC) is estimated to be the third leading cause of cancer death in the United States [1] and is projected to become the second cause of cancer-related death by 2030 [2]. PDAC is one of the cancers with the lowest survival rate (7% in 5 years), due to a late diagnosis and the intrinsic aggressiveness of this tumour [3]. Unfortunately, and in contrast to the declining trends for most cancers, PDAC is one of the few neoplasms with increasing incidence and mortality in both genders [1, 4]. Despite the efforts made in the last decades to discover new biomarkers, there is still a lack of biomarkers with enough specificity and sensitivity. This reduces the chances of detection of PDAC in early stages, when curative procedures are most effective [5]. For the majority of patients the only available treatment is the administration of gemcitabine, a chemotherapeutic agent that has shown beneficial effects in only 20-30% of patients [6, 7]. Mucins are high molecular weight glycoproteins expressed on epithelial cells. A multitude of oligosaccharides are attached to the polypeptide backbone of mucins which can be bound to the cell membrane or secreted into the organ lumen. Mucins constitute a selective barrier and play an important role in cell adhesion, immune response, cell signalling and in the renewal and differentiation of the epithelium [8-10]. Mucin expression and their glycosylation are altered in many tumours [11, 12], including breast [13], colon [14, 15], gastric [16-18], lung [19], and PDAC [20, 21]. These alterations influence cellular growth, differentiation, malignant transformation, adhesion, invasion and immune surveillance [8, 11, 22, 23]. MUC1 is a membrane-bound mucin that contains a variable number of tandem repeats (VNTR), between 20 and 125, which are abundantly O-glycosylated. Its molecular mass is therefore variable ranging from 120-225 kDa (protein backbone) and significantly larger when glycosylated, up to 500 kDa. MUC1 can also be secreted after cleavage of its extracellular domain. It is expressed at the apical surface of ductal epithelia of tissues, including breast, pancreas, bronchus and the gastrointestinal tract [17, 24, 25]. In normal pancreas, the common staining of MUC1 is at the cytoplasmic level in acinar cells and in the apical area of ductal cells [26], and it is well-established that MUC1 is overexpressed in malignant situation [26, 27]. MUC5AC is a secreted mucin of a polypeptide chain of approximately 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 586 kDa, but with a much higher molecular weight especially due to the large number of Oglycosylated chains attached to Ser and Thr residues in the VNTR regions. It is only expressed in 24% of healthy pancreas [6]. Regarding PDAC, MUC5AC has been shown to be expressed in the cytoplasm and is de novo expressed in around 75-92% of the PDAC cases [27-29] and is also detected in 70% of PanIN1A cases, which is the earliest stage of the most common precursor lesions of PDAC [27]. Aberrant glycosylation of cell surface glycolipids, membrane associated glycoproteins and secreted glycoproteins is a universal feature of malignant transformation [23, 30-32]. For instance, in malignant pancreatic tissues the carbohydrate antigens sialyl-Lewis x (SLex) and sialyl Tn (STn) have been described to be commonly expressed over 80% [33, 34] and 77% [27] respectively, while they are not detected in healthy pancreatic specimens [33, 35]. Moreover, SLex and STn expression have been associated with tumour progression and metastasis [36, 37]. STn is an O-linked disaccharide which consists of an α2,6-linked N-acetylneuraminic acid attached to N-acetylgalactosamine and is often aberrantly expressed in the neoplastic lesions including PDAC [32]. SLex epitope is a terminal structure (N-acetylneuraminic acid α2,3-Galactoseβ1,4[Fucose α1,3]N-acetylglucosamine) that can be found in either Nor O-glycosylated molecules [32, 38]. Mucins are known to be major carriers of tumour associated carbohydrate antigens and, since they are overexpressed in adenocarcinomas, these glycan alterations are highly amplified on the surface of tumour cells [12]. In gastric tissues, a recent study that characterizes mucin O-glycosylation, reports the presence of sialylated glycans, including sialyl-Lewis antigens. A higher level of sialylation and sulfation on gastric O-glycans were found in cancerous tissue compared to healthy tissue [18]. In colon cancer, an increase expression of a core 3 sialyl-Lex hexasaccharide, which appeared to compete with its sulfo-Lex counterpart in normal tissue, was described on MUC2, suggesting its use as a potential marker of malignant transformation [15]. Therefore, the detection of the cancer related glycosylation on mucins could give rise to more specific and /or sensitive cancer diagnostic markers. The expression of STn antigen on MUC1 and MUC5AC has been previously described in stomach, colon, lung, breast and ovarian tumours [39]. Recently, Remmers and coauthors have shown the expression of Tn/STn on MUC1 in PDAC tissues (91%) using an antibody directed to Tn/STn MUC1 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 glycoforms [40, 41]. The SLex epitope on MUC1 and MUC5AC has been identified in colon, gastric, ovarian and breast carcinomas [17, 39, 42-45], but to our knowledge it has not been investigated in PDAC tissues. Here we have used the in situ proximity ligation assay (PLA) to evaluate the glycan epitopes SLex and STn on MUC1 and MUC5AC mucins in a cohort of PDAC tissues. In situ PLA provides information of two epitopes in close proximity and therefore has been used to report the colocalization of two antigens indicating, in our case, the expression of a particular glycan epitope on a specific mucin. Our results showed that the expression of SLex on MUC1 and MUC5AC was observed in 68% and 84% of the mucin expressing PDAC tissues, respectively. In a second approach, PDAC tissues were assayed for MUC5AC and SLex by western blotting and 75% of them showed positivity for both markers. An immunoprecipitation protocol using specific antibodies against MUC5AC was set up. MUC5AC was immunocaptured in selected PDAC tissues that were individually positive for MUC5AC and SLex. These immunoprecipitated samples were then probed for SLex, and showed that the SLex epitope was present on MUC5AC. 2. Materials and methods 2.1 Human tissue samples Pancreatic tissues were obtained from the Hospital Dr. Josep Trueta (Girona, Spain) from patients undergoing surgical resection following the standard operating procedures of its Ethics Committee. Normal pancreatic tissue refers to adjacent, non-tumour pancreatic tissue from patients with pancreatic-related disorders. The histopathologic features of the resected specimens were confirmed by biopsy or image examination by the Digestive and Pathology Units and classified according to the Tumour Node Metastasis Classification of Malignant Tumours of the International Union Against Cancer (UICC) 7th edition [46]. For immunohistochemistry and PLA, 21 PDAC and three normal tissues were used. PDAC tissues were from 15 males and 6 females, ranging 49–81 years old; six were stage IIA (1 well differentiated, 4 moderately differentiated and one poorly differentiated), thirteen stage IIB (five well differentiated, six moderately differentiated and two poorly differentiated), one stage II* (unclassified A or B due to the lack of available information of the nodule affectation, 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 moderately differentiated), and one stage IV (well differentiated). Tissues were fixed in 10% formalin, embedded in paraffin and cut into 5 µm serial sections for immunohistochemistry (IHC) and in situ Proximity Ligation Assays (PLAs). Hematoxilin eosin staining of the tissues was performed in the same tissue blocks but not in the consecutive serial slides of the IHC and PLA assays. For protein lysates, eight PDAC and four normal tissues were used. PDAC tissues were from seven males and one female; ranging 59-79 years old. Six tissues were stage IIB and two stage IV. A small piece of each tissue was immediately frozen in liquid nitrogen and kept at -80ºC until their use. 2.2 Immunohistochemistry Paraffin sections were dewaxed and rehydrated. Endogenous peroxidase activity was blocked with 3% H2O2 in methanol for 10 min, and then sections were incubated with normal rabbit serum diluted 1/5 in PBS containing 10% BSA. Normal serum excess was removed and replaced by specific primary antibody in the appropriate concentration (Table 1) in PBS with 5% BSA and incubate overnight at 4 °C. Slides were then washed in PBS and incubated for 30 min with secondary biotinylated rabbit antimouse antibody (Dako, Glostrup, Denmark) diluted 1/200 in PBS containing 5% of BSA. The slides were subsequently washed in PBS and incubated for 30 min with avidin-biotin complex (Vectastain Elite ABC kit, Burlingame, CA) according to the manufacturer’s recommendations. Staining was performed with 3,3-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO) containing 0.02% hydrogen peroxide. Counterstaining of the nucleus was done with Mayer’s hematoxylin solution. Slides were examined under a bright-field microscope AH2 Vanox-T microscope Olympus. Images were acquired using an Olympus microscope digital camera DP73 and the CellSens Standard 1.11 software. SLex and MUC1-M8 immunostainings were performed using the KM93 and M8 monoclonal detection antibodies, and a biotinylated goat anti-mouse IgM at 1/200 and a biotinylated horse anti-IgG at 1/100 antibodies (Vectastain Elite ABC kit) in 1.5% of goat serum or 1.5% of horse serum, respectively in PBS, following the protocol described in [47]. CSLEX antibody (at 1/20 dilution) (BD Biosciences, San Jose, CA) was used to compare the SLex immunostaining obtained with the KM93 antibody. 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 Staining intensity was graded to the frequency of positive cells as: strongly positive (>60% stained cells), moderately positive (25-60% stained cells), faintly positive (<25% stained cells), sparse cells and negative. The histological analysis and scoring of the tissues were performed by two independent expert pathologists of the Hospital Dr. Josep Trueta of Girona. 2.3 Purification and quantification of monoclonal antibodies VU4H5 (MAb anti-MUC1), CLH2 (MAb anti-MUC5AC) and TKH2 (MAb anti-STn) were purified with HiTrap Protein G sepharose columns (GE Healthcare, Amersham Biosciences, Üppsala, Sweden), according to the manufacturer’s protocol. Desorption buffer was exchanged into phosphate buffer saline (PBS, pH 7.4) using Amicon Ultra-0.5 10K Centrifugal Filters (Millipore, Billerica, MA). 2.4 In situ Proximity Ligation Assay In situ Proximity Ligation Assay (PLA) was performed in paraffin sections from controls and PDAC cases for colocalization analysis of SLex and STn antigens with the MUC1 and MUC5AC mucins. Firstly, Duolink In Situ Probemaker kit (Sigma-Aldrich, St. Louis, MO) was used to conjugate the PLA oligonucleotide arms directly to the purified primary antibodies. TKH2 (anti-STn) was conjugated with the MINUS probes (Olink DUO920010, Olink Bioscience, Uppsala, Sweden), and VU4H5 (anti-MUC1) and CLH2 (anti-MUC5AC) with the PLUS probes (Olink DUO92009) according to the manufacturer’s instructions. KM93 (MAb anti-SLex) antibody was not labelled with PLA probe due to its IgM nature. Thus, the secondary antibodies AffiniPure goat anti-mouse IgG (subclass 1) and AffiniPure goat anti-mouse IgM (both from Jackson ImmunoResearch, West Grove, PA) were labelled using the previously detailed oligonucleotides Olink DUO920010 and Olink DUO92009, respectively. Deparaffined tissue sections were pre-treated according to the immunohistochemistry protocol explained above. Then, sections were incubated for 30 min at 37ºC with the Blocking Solution (Duolink In Situ Probemaker kit). For colocalization of mucins with STn epitope, primary antibodies conjugated with oligoprobes were used. Conjugated MAbs to MUC1 (1/40) or MUC5AC (1/100) and 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 STn (1/40) were diluted in PBS with 5% BSA containing 1/20 of assay reagent (Duolink In Situ Probemaker kit) and slides were incubated overnight at 4 ºC. Slides were then washed with a filtered solution of 0.01 M Tris, 0.15 M NaCl and 0.05% Tween 20 (Wash buffer A) prior to the ligation step. The ligation solution was prepared diluting the two oligonucleotides 1/5 and the ligase 1/40 (Duolink In Situ Detection Reagents Orange, Sigma-Aldrich) in pure water, and was added to slides and incubated for 30 minutes at 37 ºC in order to hybridize with the PLA probes and to form a closed circle. After a wash with buffer A, the amplification was performed in darkness adding the amplification solution to the samples, consisting of nucleotides and fluorescently labelled oligonucleotides, together with the polymerase both diluted in pure water 1/5 and 1/80, respectively. The slides were washed with 0.2 M Tris and 0.1 M NaCl (Wash buffer B) and then were incubated with DAPI to visualise nuclei (Sigma-Aldrich, 0.4 mg/mL). After washing slides with 0.01x wash buffer B, samples were mounted with Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA). All slides were examined under a Zeiss Imager.Z1 Axio fluorescence microscope (Zeiss, Welwyn Garden City, UK). Images were acquired using a Zeiss Axio cam MRm and the AxioVision Release 4.8.1 software. The analyses of the colocalization of MUC1 and MUC5AC with SLex epitope were performed using secondary antibodies with oligoprobes. The procedure was similar to as previously described for the PLA with incubations of VU4H5, CLH2 (both IgGs subtypes) and KM93 (IgM subtype) primary antibodies (same conditions as for the immunohistochemistry) followed by 1h incubation at 37 ºC with the goat anti-mouse IgG1 and goat anti-mouse IgM oligoprobe secondary antibodies, diluted 1/300. Imaging analysis of the PLA signal was performed with the Duolink Image Tool software (SigmaAldrich). These quantifications were performed for the imaging analysis of the PLA results of SLexMUC1 and SLex-MUC5AC colocalization for each of the tissues. Briefly, several representative images per case were subjected to the software analysis and the median of positive blobs (positive PLA events) was set up for each case. Then, the median of positivity for each PLA combination was determined and 3 categories were established as follows: +++ (positive cases whose median is higher than 75% of the overall median); ++ (positive cases higher than 25% and lower than 75%); and + (positive cases lower than 25%). 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 2.5 Tissue protein lysate Frozen PDAC and normal pancreatic tissues were thawed, mixed with RIPA buffer [(20 mM sodium phosphate, 150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100 (pH 7.4,)] supplemented with a protease inhibitor cocktail Complete Ultra tablets (Roche, Basel, Switzerland), and homogenised with lysing matrix beads D in a FastPrep-24 Instrument (MP Biomedicals, Santa Ana, CA). Tissue homogenates were centrifuged 5 min at 14000g at 4 ºC. The supernatant was collected and total protein quantified with bicinchoninic acid assay (BCA) (Thermo Scientific Pierce, Waltham, MA). 2.6 Immunoprecipitation For immunoprecipitation assays, cell lysates containing equal amounts of protein (500 µg) were reduced in buffer containing dithiothreitol such that the final concentration when mixed with sample was 10 mM. After 2 h incubation at 37 ºC, fresh iodoacetamide was added to a final concentration of 25 mM and samples were incubated at room temperature for 30 minutes in the dark. After reduction and alkylation, samples were desalted and concentrated using Amicon Ultra-0.5 3kDa Centrifugal Filter Devices (Millipore) which had been previously passivated with 5% Brij-35 (Sigma). Next, samples were diluted in incubation buffer,50 mM Tris pH 7.4, 150 mM NaCl and 1% Triton X100, 5mM EDTA and protease inhibitors (complete ultratablets from Roche), and precleared with 100 µL of Protein G agarose resin for two hours at 4 ºC. The precleared samples were incubated with 20 µL of anti-MUC5AC polyclonal antibody (Lum5.1) for two hours at 4 ºC. The immunocomplexes were then captured by incubating samples with Protein G agarose resin packed into a spin column (Corning Costar Spin-X, New York, NY) overnight at 4 ºC. Protein G column were centrifuged and washed three times with washing buffer (50 mM Tris pH 7.4, 150 mM NaCl and 1% Triton X-100). MUC5AC bound to the Lum5.1 antibody was eluted after boiling the sample with Laemmli buffer containing 1.25% β-mercaptoethanol. 2.7 Western Blot 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 been previously described by other authors in gastric, colon, lung, breast and ovarian cancer tissues [17, 39, 42-45] as well as in pancreatic cancer cell line [71], using PLA and other antibody-based approaches. Since an increase of truncated structures is a key feature of tumour cells [72], we expected an increase in STn expression on MUC1 and MUC5AC. Although the expression of MUC1 bearing Tn/STn, detected by a specific anti-MUC1-Tn/STn antibody, in PDAC tissues has been reported by Remmers and collaborators [40], our results do not show the same tendency. This could be due to the use of different antibodies, different methodology and different cohort. We have used a specific antiSTn antibody and PLA technique to colocalize this antigen within the mucin proteins. Regarding the SLex epitope on these mucins, our results show a wide colocalization of SLex on MUC1 and especially on MUC5AC in PDAC tissues. Furthermore, the presence of SLex on MUC5AC was confirmed by immunoprecipitation of PDAC tissue protein lysates and subsequent Western blotting analysis using anti-SLex antibodies. Our results show that both mucins are carriers of SLex antigen in most PDAC tissues that expressed individually SLex and the corresponding mucin. However, other glycoconjugates could also be carriers of SLex antigen. Most of the currently used cancer biomarkers assays take advantage of changes in the biodistribution of tumour cell derived products [12, 20, 32], such as the diagnostic serum tests that are based on changes in the level of the circulating mucins, MUC16 (CA125) [73, 74] and MUC1 (CA15-3) [75], or changes in the mucin specific terminal glycan structures sialyl-Lewis a (CA19-9)[76, 77] or STn (CA72-4) [78, 79]. To date, there is no specific tumour marker to diagnose PDAC. The only marker approved by FDA to manage PDAC is CA19-9 that detects sialyl-Lewis a, an epitope found mainly on mucins [80]. Some mucin-based markers lack specificity due to their detection also in benign conditions. Glycan-based markers, on the other hand, present low tissue specificity since aberrant Oglycans can be found in many carcinoma tissues. Therefore, our strategy was the detection of a specific glycoform on a specific mucin that could circumvent these weaknesses and generate a biomarker with potential application for specific PDAC detection. A good biomarker should be a molecule that can be found in blood or other fluids. However, due to the pathophysiology and micro-architecture of PDAC, which is poorly perfused and vascularized, secreted proteins that could be candidates for biomarkers have rarely been detected in circulation [81]. 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 On the other hand, PDAC tissue contains higher concentration of PDAC-specific markers and is the most direct source for the finding of cancer biomarkers [82, 83]. Thus, our strategy has been to analyse pancreatic tissues to identify candidates for their future validation in fluids, such as serum of PDAC patients. In the present study, the colocalization of MUC1 and MUC5AC with the tumour epitope SLex presented a high specificity due to SLex absence in non-malignant control tissue and has potential to be considered a PDAC biomarker. In this regard, further analyses of the glycan epitope SLex on MUC1 and MUC5AC in serum from PDAC patients are warranted, particularly the MUC5AC-SLex glycoform that showed 84% of expression in the MUC5AC expressing PDAC tumours, including poorly, moderately and highly differentiated tissues. Conclusions In this study, we have evaluated the anti-MUC1 VU4H5 antibody in pancreatic tissues and we have also studied the colocalization of the tumour associated antigens SLex and STn within the mucins MUC1 and MUC5AC in pancreatic cancer tissues. We have shown a wide expression of SLex on MUC1 and MUC5AC in the tumour cells and in the secretions. We have confirmed, by immunoprecipitation of MUC5AC followed by SLex probing, the presence of SLex on MUC5AC in PDAC tissues. Since mucins can reach the bloodstream and be detected in fluids such as pancreatic juice or serum, the herein presented results suggest that the glycoforms MUC1-SLex and MUC5ACSLex could be good candidates for pancreatic cancer serum biomarkers. Acknowledgements M.B. acknowledges University of Girona for a pre-doctoral fellowship and a mobility grant. We also thank David Carreras for part of the IHC analysis. The authors also thank Dr. Carme de Bolós from Gastroesophagic Cancer Research Group, Hospital del Mar Medical Research Institute (IMIM), for kindly providing anti-MUC5AC rabbit polyclonal antibody Lum5.1. This work was supported by Spanish Ministry of Science and Innovation (grant BIO 2015-66356-R, awarded to R.P.). 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Hakomori, Preparation and characterization of monoclonal antibodies directed to the tumor-associated O-linked sialosyl-2----6 alpha-N-acetylgalactosaminyl (sialosyl-Tn) epitope, Cancer Res 48(8) (1988) 2214-20. 1240 1241 1242 1243 1244 1245 1246 1247 1248 1249 1250 1251 1252 1253 1254 1255 1256 1257 1258 1259 1260 1261 1262 1263 1264 1265 1266 1267 1268 1269 1270 1271 1272 1273 1274 1275 1276 1277 1278 1279 1280 1281 1282 1283 1284 1285 1286 1287 1288 1289 1290 1291 1292 1293 1294 1295 1296 1297 1298 Table 1: Antibodies used for immunohistochemistry and Western blotting. Antibody Isotype Concentration Antigen and reference CLH2 IgG2 0.60 ng/µL MUC5AC [84] KM93 IgM 2.5 ng/µL IHC/PLA 3.3 ng/µL western blotting SLex (Merck Millipore, Darmstadt, Germany) LUM5.1 polyclonal Rabbit polyclonal serum diluted 1/1000 western blotting MUC5AC [85] M8 IgG1 Supernatant diluted 1/2 MUC1 [86-88] TKH2 IgG1 0.95 ng/µL STn [89] VU4H5 IgG1 1.19 ng/µL MUC1 [60-62] 1299 1300 1301 1302 1303 1304 1305 1306 1307 1308 1309 1310 1311 1312 1313 1314 1315 1316 1317 1318 1319 1320 1321 1322 1323 1324 1325 1326 1327 1328 1329 1330 1331 1332 1333 1334 1335 1336 1337 1338 1339 1340 1341 1342 1343 1344 1345 1346 1347 1348 1349 1350 1351 1352 1353 1354 1355 1356 1357 Table 2: Carbohydrate epitopes SLex and STn expression in pancreatic tissues. The stage II* refers to a patient that the N (regional lymph nodes) cannot be addressed. PDAC category and stage (TNM system) Normal pancreas Stage IIA (T3N0M0) Stage IIB (T3N1M0) Stage II* (T3NxM0) Stage IV (T3N1M1) negative 3/3 (100%) - - - - sparse cells - - - - - faintly positive - - 1/13 (8%) 1/1 (100%) - moderately positive - 5/6 (83%) 7/13 (54%) - - SLex strongly positive - 1/6 (17%) 5/13 (38%) - 1/1 (100%) negative 3/3 (100%) 1/6 (17%) 2/13 (15%) - - sparse cells - - 2/13 (15%) - - faintly positive - 4/6 (67%) - - - moderately positive - 1/6 (17%) 6/13 (46%) - 1/1 (100%) STn strongly positive - - 3/13 (23%) 1/1 (100%) - 1358 1359 1360 1361 1362 1363 1364 1365 1366 1367 1368 1369 1370 1371 1372 1373 1374 1375 1376 1377 1378 1379 1380 1381 1382 1383 1384 1385 1386 1387 1388 1389 1390 1391 1392 1393 1394 1395 1396 1397 1398 1399 1400 1401 1402 1403 1404 1405 1406 1407 1408 1409 1410 1411 1412 1413 1414 1415 1416 Table 3. Carbohydrate epitopes SLex and STn and mucins MUC1 and MUC5AC expression in each of the pancreatic tissues analysed. Category and stage (TNM system) Cases Differentiation grade1 Gender Age SLex STn MUC1 MUC5AC - M 65 - - +++ - - F 76 - - +++ - Normal Pancreas 3 - F 61 - - +++ - MD M 70 ++ + +++ +++ MD M 65 ++ + ++ ++ MD M 61 ++ ++ ++ ++ PD M 65 ++ ++ +++ + WD/MD M 71 +++ - sc - Stage IIA (T3N0M0) 6 MD/PD M 70 ++ + + + MD M 57 ++ ++ ++ + MD M 72 +++ ++ +++ ++ WD F 70 ++ +++ ++ +++ PD M 58 ++ ++ +++ ++ WD M 72 +++ ++ ++ + WD F 49 +++ - + - WD F 78 +++ +++ + +++ PD M 81 ++ sc +++ + MD F 71 ++ ++ sc ++ MD M 67 ++ ++ +++ ++ WD/MD F 66 ++ - ++ ++ MD F 57 + sc ++ + Stage IIB (T3N1M0) 13 MD M 79 ++ +++ +++ +++ Stage II* (T3NxM0) 1 MD M 65 + +++ +++ +++ PDAC Stage IV (T3N1M1) 1 WD M 76 +++ ++ +++ +++ Staining intensity is graded as: negative: -, sparse cells: sc; faintly positive (10-25% stained cells): +; moderately positive (25-60% stained cells): ++; and strongly positive (>60% stained cells): +++. The stage II* refers to a patient that the N (regional lymph nodes) cannot be addressed. 1Differentiation grade: PD: poorly differentiated, MD: moderately differentiated, and WD: well differentiated. 1417 1418 1419 1420 1421 1422 1423 1424 1425 1426 1427 1428 1429 1430 1431 1432 1433 1434 1435 1436 1437 1438 1439 1440 1441 1442 1443 1444 1445 1446 1447 1448 1449 1450 1451 1452 1453 1454 1455 1456 1457 1458 1459 1460 1461 1462 1463 1464 1465 1466 1467 1468 1469 1470 1471 1472 1473 1474 1475 Supplementary figures Figure S1. SLex staining pattern using KM93 and CSLEX antibodies. Immunohistochemistry staining of the SLex epitope at different magnification (x40 and x200) of two representative pancreatic adenocarcinoma tissues. A-D corresponds to a IIB, moderately differentiated PDAC (PDAC A) and E-H corresponds to a IIB, well differentiated PDAC (PDAC B) Figure S2: Expression pattern of MUC5AC, MUC1, SLex, STn and vinculin in six of the the pancreatic tissue lysates. Western blots of 20 µg of one control (C1) and of five PDAC tissue lysates (T1, T4, T5, T7, T8) to detect mucins (MUC5AC and MUC1), carbohydrate antigens (SLex and STn) and vinculin.