_:b546878540 . _:b29436437 . . _:b546878344 "4"^^ . _:b546878541 . _:b546878542 . _:b546878734 . _:b546878327 . _:b546878543 . _:b546878536 . _:b29436438 . _:b546878347 "3"^^ . _:b546878350 . _:b29436448 "and, in normoxic conditions, catalyzes the hydroxylation of proline residues of the hypoxia-inducible factor 1 \u03B1 (HIF1\u03B1), rendering it susceptible to poly-ubiquitinylation and subsequent proteasomal degradation (Berra et al., >>2003<<). Klotsche-van Ameln et al. have reported that inhibiting EGLN1 had an anti-tumor effect by enhancing TGF\u03B2 anti-proliferative effect in a model of murine osteosarcoma (Klotzsche-von Ameln et al., 2011)." . _:b29436437 . _:b546878712 . _:b546878537 . _:b546878538 . _:b546878729 . _:b546878326 . _:b546878539 . _:b546878532 . _:b29436439 . _:b546878533 . _:b546878346 "4"^^ . _:b546878534 . _:b546878728 . _:b546878321 . _:b546878535 . _:b546878528 . _:b29436432 . _:b546878357 "3"^^ . _:b546878529 . _:b29436398 "In the context of breast cancer, recent studies have shown that the cross-talk and interaction of the tumor cells with their surrounding microenvironment is necessary for tumor progression (Tlsty and Coussens, 2006; Polyak et al., >>2009<<; Dvorak et al., 2011; Boudreau et al., 2012; Conklin and Keely, 2012; Fordyce et al., 2012)." . _:b546878351 . _:b546878731 . _:b546878530 . . _:b546878320 . _:b546878531 . _:b546878652 . _:b29436433 . _:b546878356 "3"^^ . . _:b546878472 . _:b546878653 . _:b546878654 . _:b546878730 . _:b546878323 . _:b546878655 . _:b546878648 . _:b29436434 . _:b546878359 "3"^^ . _:b546878649 . . _:b546878741 . _:b546878650 . _:b546878322 . _:b546878651 . . _:b29436435 . _:b546878358 "3"^^ . _:b546878644 . _:b546878645 . _:b546878646 . _:b546878740 . _:b546878333 . _:b546878647 . _:b546878640 . _:b29436444 . _:b546878353 "3"^^ . . _:b546878690 . _:b546878641 . _:b546878642 . _:b546878332 . _:b546878696 . _:b546878643 . _:b546878636 . _:b29436445 . _:b546878352 "3"^^ . . _:b546878637 . . _:b546878742 . _:b546878638 . _:b546878335 . _:b546878551 . _:b546878639 . _:b546878632 . _:b29436446 . _:b546878355 "3"^^ . _:b546878633 . _:b546878627 . _:b546878634 . _:b546878737 . _:b546878334 . _:b546878635 . . _:b29436447 . _:b546878354 "3"^^ . _:b546878628 . _:b546878629 . . _:b546878736 . _:b546878630 . _:b546878329 . _:b546878631 . _:b546878624 . _:b546878365 "3"^^ . . _:b546878625 . _:b29436440 . _:b546878626 . _:b29436396 . _:b546878739 . _:b546878328 . _:b546878466 . _:b546878627 . _:b546878620 . _:b29436441 . _:b546878491 . _:b546878364 "3"^^ . . _:b546878621 . . _:b546878738 . _:b546878622 . _:b546878331 . _:b546878623 . _:b546878616 . _:b29436442 . _:b546878367 "3"^^ . _:b546878617 . _:b29436413 _:b29436416 . _:b546878642 . _:b546878618 . _:b546878330 . _:b546878619 . _:b29436413 _:b29436417 . _:b29436413 _:b29436418 . _:b29436443 . _:b29436410 "Pathologists have used excessive ECM deposition (desmoplasia) as a marker of tumors with poor prognosis long before the complexity of the ECM was even deciphered (Anastassiades and Pryce, >>1974<<). Despite its great importance in physiological (development, aging) and pathological processes such as cancer, the extracellular matrix remains underexplored (Wilson, 2010)." . _:b546878366 "3"^^ . _:b546878612 . _:b29436413 _:b29436419 . _:b546878613 . _:b29436413 _:b29436420 . _:b546878614 . _:b546878341 . _:b546878615 . _:b29436413 _:b29436421 . _:b546878608 . _:b29436452 . _:b546878361 "3"^^ . _:b546878609 . _:b546878610 . _:b546878340 . _:b546878611 . . _:b29436453 . _:b546878360 "3"^^ . _:b546878604 . . _:b546878605 . _:b546878376 . . _:b546878606 . _:b546878343 . _:b29436426 "According to our previous bioinformatic definition of the matrisome (Naba et al., >>2012<<), we further categorized these ECM proteins into two categories:" . _:b546878607 . _:b546878600 . _:b29436454 . _:b546878363 "3"^^ . _:b546878601 . _:b29436451 . _:b546878602 . _:b546878342 . _:b546878603 . _:b546878596 . _:b29436455 . _:b546878362 "3"^^ . _:b546878597 . . _:b546878598 . _:b546878337 . _:b546878599 . . _:b546878592 . _:b29436448 . _:b546878547 . _:b546878373 "3"^^ . _:b546878593 . _:b546878594 . _:b546878336 . _:b546878595 . . _:b546878716 . _:b29436449 . _:b546878372 "3"^^ . _:b546878717 . _:b546878718 . _:b546878400 . _:b546878339 . _:b546878719 . _:b546878712 . _:b29436450 . _:b546878375 "3"^^ . _:b546878713 . _:b546878715 . _:b546878714 . _:b546878338 . _:b546878715 . _:b546878414 . _:b546878708 . _:b29436451 . _:b546878374 "3"^^ . _:b546878709 . _:b546878710 . _:b546878349 . _:b546878711 . _:b546878704 . _:b546878369 "3"^^ . _:b546878705 . _:b546878594 . _:b546878706 . _:b546878348 . _:b546878707 . _:b546878700 . _:b546878633 . _:b546878368 "3"^^ . _:b546878701 . _:b546878702 . _:b546878351 . _:b546878703 . _:b546878696 . _:b546878371 "3"^^ . _:b546878697 . _:b546878698 . _:b546878585 . _:b546878350 . _:b546878699 . _:b546878692 . _:b546878370 "3"^^ . _:b546878693 . _:b546878694 . _:b546878345 . _:b546878695 . . _:b29436456 . _:b546878381 "3"^^ . _:b546878688 . _:b546878689 . . _:b546878690 . _:b546878344 . _:b546878691 . _:b546878684 . _:b29436457 . _:b546878380 "3"^^ . _:b546878685 . . _:b29436436 "We also performed RT-qPCR analyses for several other proteins in our lists despite the fact that it is well known that mRNA levels and steady-state protein levels do not correlate at all well (Gygi et al., >>1999<<). It can be seen that the mRNA changes for several of the proteins of interest (CYR61, EGLN1, LTBP3) do not show mRNA changes corresponding with the marked differences in protein levels detected, although some do (S100A2, SNED1) (Figure" . _:b546878686 . _:b546878347 . _:b546878687 . _:b546878383 "3"^^ . _:b29436458 . _:b546878680 . . _:b546878681 . _:b546878682 . _:b29436437 "LM2 cells have been selected for their high metastatic potential and their tropism for the lungs (Minn et al., >>2005<<). In our system, LM2 cells are injected in the absence of exogenous ECM support (Matrigel) in immunodeficient mice (NOD/SCID/IL2R\u03B3-null)." . _:b546878346 . _:b546878683 . _:b546878382 "3"^^ . _:b546878676 . _:b546878677 . _:b546878678 . _:b546878357 . _:b546878679 . _:b546878377 "3"^^ . _:b546878672 . _:b546878673 . _:b546878674 . _:b546878632 . _:b546878356 . _:b546878675 . _:b546878376 "3"^^ . _:b546878668 . _:b546878405 . . . _:b546878669 . . _:b546878670 . _:b546878359 . _:b546878671 . "10.7554%2FeLife.01308.024" . _:b546878379 "3"^^ . _:b546878664 . _:b546878665 . _:b546878666 . _:b546878358 . _:b546878667 . _:b546878378 "3"^^ . _:b546878660 . . . _:b546878661 . . _:b546878445 . _:b546878662 . _:b546878353 . _:b546878663 . . . _:b546878389 "3"^^ . _:b546878656 . _:b546878413 . _:b546878657 . . _:b546878658 . _:b546878352 . _:b546878659 . _:b546878388 "3"^^ . _:b546878348 . _:b546878355 . _:b546878391 "3"^^ . _:b546878349 . _:b546878640 . . _:b546878354 . _:b546878390 "3"^^ . . _:b546878365 . _:b546878644 . _:b546878385 "3"^^ . . _:b546878370 . _:b546878364 . _:b546878384 "3"^^ . _:b546878367 . _:b546878582 . . _:b546878387 "3"^^ . _:b546878366 . _:b546878386 "3"^^ . _:b546878677 . _:b546878361 . _:b546878397 "3"^^ . . _:b546878614 . _:b546878360 . _:b546878396 "3"^^ . _:b29436435 . . _:b546878363 . _:b546878399 "3"^^ . _:b546878362 . _:b546878740 . . . _:b546878398 "3"^^ . _:b546878741 . _:b546878742 . _:b546878373 . _:b546878736 . _:b546878393 "3"^^ . _:b546878737 . _:b546878738 . _:b546878739 . _:b546878372 . _:b546878732 . _:b546878392 "3"^^ . _:b546878733 . _:b546878734 . _:b546878486 . . _:b546878735 . _:b546878375 . _:b546878297 . _:b546878395 "3"^^ . _:b546878728 . _:b546878729 . . _:b546878730 . _:b546878731 . _:b546878374 . _:b546878724 . _:b546878394 "3"^^ . _:b546878725 . _:b29436449 . _:b546878726 . _:b546878727 . . . _:b546878369 . _:b546878720 . _:b546878405 "3"^^ . _:b546878721 . . _:b546878722 . _:b546878723 . _:b546878368 . _:b546878404 "3"^^ . _:b29436400 "studies have shown that the cross-talk and interaction of the tumor cells with their surrounding microenvironment is necessary for tumor progression (Tlsty and Coussens, 2006; Polyak et al., 2009; Dvorak et al., 2011; Boudreau et al., >>2012<<; Conklin and Keely, 2012; Fordyce et al., 2012)." . _:b546878345 . _:b546878371 . _:b546878407 "2"^^ . _:b29436413 "materials and methods" . _:b546878370 . _:b546878406 "3"^^ . . _:b546878554 . _:b546878381 . _:b546878401 "3"^^ . _:b29436431 . _:b546878380 . _:b546878400 "3"^^ . _:b546878732 . _:b546878383 . _:b546878403 "3"^^ . _:b546878382 . _:b546878402 "3"^^ . _:b546878411 . . _:b546878413 "2"^^ . _:b546878377 . _:b546878412 "2"^^ . _:b546878622 . _:b546878452 . _:b546878376 . _:b546878649 . _:b546878415 "2"^^ . _:b546878379 . _:b546878537 . _:b546878414 "2"^^ . _:b29436457 "interactions between ECM proteins and their cellular receptors (Goodman and Picard, 2012) or by disrupting the architecture of the ECM (that acts as a barrier) to allow more efficient drug delivery (Yuan et al., 2007; Whatcott et al., >>2011<<; Jacobetz et al., 2013), the proteins discovered in our differential proteomics-based approach have the potential to serve as novel anticancer therapeutic targets." . _:b546878623 . _:b546878378 . . _:b546878409 "2"^^ . _:b546878389 . _:b546878570 . _:b546878408 "2"^^ . _:b546878388 . _:b546878488 . _:b546878411 "2"^^ . _:b546878729 . _:b546878322 . _:b546878391 . . . _:b546878410 "2"^^ . _:b546878390 . _:b546878596 . _:b546878421 "2"^^ . _:b546878385 . . _:b546878741 . _:b546878500 . _:b546878420 "2"^^ . . _:b546878384 . _:b29436433 "A strength of human/mouse xenograft model systems coupled to mass spectrometry is that they allow the distinction of human (tumor-derived) protein sequences from their murine (stroma-derived) counterparts (Naba et al., >>2012<<). Therefore we could determine the relative contributions of tumor and stromal cells to the production of the tumor ECM." . _:b546878364 . _:b546878423 "2"^^ . _:b546878387 . . _:b546878422 "2"^^ . _:b546878386 . . _:b546878661 . _:b546878673 . _:b546878663 . . _:b546878417 "2"^^ . _:b546878397 . _:b546878316 . _:b546878504 . _:b546878416 "2"^^ . _:b546878522 . _:b546878396 . . _:b546878336 . . _:b546878419 "2"^^ . . _:b546878399 . _:b546878442 . _:b546878578 . _:b546878418 "2"^^ . _:b546878398 . _:b29436446 . . _:b546878429 "2"^^ . _:b546878484 . _:b546878393 . _:b546878421 . _:b546878301 . _:b546878560 . _:b546878428 "2"^^ . _:b546878669 . _:b546878392 . . _:b546878693 . _:b546878328 . _:b546878431 "2"^^ . _:b546878395 . _:b546878447 . _:b546878430 "2"^^ . _:b546878383 . _:b546878394 . _:b546878604 . . _:b546878425 "2"^^ . _:b546878405 . . _:b546878424 "2"^^ . _:b546878404 . . . . _:b546878427 "2"^^ . _:b29436432 "Examples of these are angiopoietin-like 4 (ANGPTL4, Padua et al., 2008), cathepsin B (CTSB, Sevenich et al., 2010, 2011; Vasiljeva et al., 2006), insulin-like growth factor-binding protein 4 (IGFBP4, Ryan et al., >>2009<<) and lysyl-oxidase-like 2 (LOXL2, Barry-Hamilton et al., 2010)." . _:b546878407 . . . _:b546878426 "2"^^ . _:b546878406 . . . _:b546878401 . _:b546878437 "2"^^ . _:b546878400 . _:b546878706 . _:b546878436 "2"^^ . _:b29436399 "breast cancer, recent studies have shown that the cross-talk and interaction of the tumor cells with their surrounding microenvironment is necessary for tumor progression (Tlsty and Coussens, 2006; Polyak et al., 2009; Dvorak et al., >>2011<<; Boudreau et al., 2012; Conklin and Keely, 2012; Fordyce et al., 2012)." . . _:b546878403 . _:b546878439 "2"^^ . _:b546878735 . _:b546878542 . _:b546878402 . _:b546878688 . _:b546878516 . _:b546878438 "2"^^ . . _:b546878413 . _:b546878433 "2"^^ . . . _:b546878412 . _:b546878432 "2"^^ . . . _:b546878365 . . _:b546878415 . _:b546878737 . _:b546878435 "2"^^ . _:b546878414 . _:b546878434 "2"^^ . _:b546878465 . _:b546878409 . _:b546878445 "2"^^ . . _:b546878408 . _:b546878444 "2"^^ . . . . _:b546878411 . _:b546878447 "2"^^ . _:b546878535 . _:b546878410 . _:b546878446 "2"^^ . _:b546878490 . . . . _:b546878421 . _:b546878441 "2"^^ . . . _:b546878420 . _:b546878440 "2"^^ . _:b546878492 . _:b546878595 . _:b546878423 . _:b546878443 "2"^^ . _:b546878617 . _:b546878361 . _:b546878422 . _:b546878442 "2"^^ . _:b546878304 . _:b546878417 . _:b546878453 "2"^^ . _:b546878416 . _:b546878452 "2"^^ . . . _:b29436430 "Examples of these are angiopoietin-like 4 (ANGPTL4, Padua et al., 2008), cathepsin B (CTSB, Sevenich et al., 2010, >>2011<<; Vasiljeva et al., 2006), insulin-like growth factor-binding protein 4 (IGFBP4, Ryan et al., 2009) and lysyl-oxidase-like 2 (LOXL2, Barry-Hamilton et al., 2010)." . _:b546878455 "2"^^ . . _:b546878419 . . _:b546878418 . _:b546878454 "2"^^ . _:b546878346 . . . . _:b546878508 . _:b546878429 . _:b546878449 "2"^^ . . _:b546878428 . _:b546878448 "2"^^ . . _:b546878621 . _:b546878538 . _:b546878503 . _:b546878431 . _:b546878451 "2"^^ . _:b546878352 . _:b546878450 "2"^^ . _:b546878430 . _:b546878425 . _:b546878461 "2"^^ . . . _:b546878676 . _:b546878460 "2"^^ . _:b546878424 . . _:b546878526 . _:b546878507 . _:b546878463 "2"^^ . _:b546878427 . . . _:b546878590 . _:b546878357 . _:b546878444 . _:b546878426 . _:b546878462 "2"^^ . . _:b546878356 . . _:b546878437 . _:b546878457 "2"^^ . _:b29436440 _:b29436444 . _:b29436440 _:b29436445 . _:b546878456 "2"^^ . _:b546878436 . . _:b29436440 _:b29436446 . _:b29436440 _:b29436447 . _:b29436440 _:b29436441 . _:b546878459 "2"^^ . _:b546878439 . _:b29436440 _:b29436442 . . _:b29436440 _:b29436443 . _:b29436440 _:b29436452 . _:b29436440 _:b29436453 . _:b546878458 "2"^^ . _:b546878438 . _:b29436440 _:b29436454 . _:b546878660 . _:b29436440 _:b29436455 . _:b29436440 _:b29436448 . _:b29436440 _:b29436449 . _:b546878433 . _:b29436440 _:b29436450 . _:b29436440 _:b29436451 . . _:b546878432 . _:b546878330 . . _:b29436440 _:b29436456 . _:b29436440 _:b29436457 . _:b546878435 . _:b29436440 _:b29436458 . _:b546878525 . _:b546878302 . _:b546878332 . _:b546878434 . . _:b546878445 . _:b546878697 . _:b546878444 . . _:b546878447 . _:b29436441 . _:b546878446 . . _:b546878441 . _:b546878493 . _:b29436418 "type=shRNA), and cloned into the MSCV-ZSG-2A-Puro-miR30 vector retroviral vector as described previously (Stern et al., 2008; Lamar et al., >>2012<<). This vector expresses the miR30-based shRNA in the 3\u2019UTR of a single transcript encoding the ZsGreen reporter gene and the puromycin-resistance gene. Retroviruses were packaged in 293FT cells and LM2 cells were transduced as previously" . . _:b546878440 . _:b29436414 "ZsGreen-positive foci were counted in the left pulmonary lobe using the open-source software Cell Profiler 2.0 (Lamprecht et al., >>2007<<; http://www.cellprofiler.org/) and counts were manually curated when needed. The Cell Profiler pipeline developed for this study is provided as a ." . _:b29436394 "decade, there has been increasing interest in the tumor microenvironment, which encompasses all the components (cellular and acellular) of a tumor in addition to the tumor cells themselves (Joyce and Pollard, 2009; Egeblad et al., >>2010<<). Indeed, in order to progress, a tumor needs to be surrounded by a permissive environment and, to metastasize, tumor cells need to find or create a favorable niche (seed and soil theory) (Paget, 1889; Ribatti et al., 2006; Erler and" . _:b546878325 . _:b29436416 . _:b546878467 . _:b546878443 . _:b546878434 . _:b546878442 . _:b29436428 "Examples of these are angiopoietin-like 4 (ANGPTL4, Padua et al., >>2008<<), cathepsin B (CTSB, Sevenich et al., 2010, 2011; Vasiljeva et al., 2006), insulin-like growth factor-binding protein 4 (IGFBP4, Ryan et al., 2009) and lysyl-oxidase-like 2 (LOXL2, Barry-Hamilton et al., 2010)." . . _:b546878453 . _:b546878579 . . _:b546878563 . _:b546878419 . _:b546878452 . _:b546878368 . _:b546878455 . _:b546878517 . . . _:b546878454 . _:b546878605 . _:b546878392 . _:b546878449 . _:b546878427 . _:b29436422 _:b29436436 . . _:b29436422 _:b29436437 . _:b546878448 . _:b29436422 _:b29436438 . . _:b546878536 . _:b546878529 . _:b29436422 _:b29436439 . . _:b546878717 . _:b29436422 _:b29436432 . _:b29436422 _:b29436433 . _:b546878451 . _:b29436422 _:b29436434 . _:b29436422 _:b29436435 . _:b29436422 _:b29436428 . _:b29436422 _:b29436429 . _:b546878450 . _:b29436422 _:b29436430 . _:b29436422 _:b29436431 . _:b29436422 _:b29436424 . _:b546878497 . _:b29436422 _:b29436425 . _:b546878461 . _:b29436422 _:b29436426 . _:b29436422 _:b29436427 . _:b29436406 . _:b546878299 . _:b546878460 . _:b29436422 _:b29436423 . _:b546878678 . . . _:b546878463 . . _:b546878672 . _:b546878462 . _:b546878654 . . _:b546878457 . . _:b546878456 . _:b29436416 "using stage tips, that is, pipette tips packed with reversed-phase membrane disks (Empore C-18 #2215, 3M Corporation, St Paul, MN), eluted with 60% acetonitrile, 0.1% TFA, and then concentrated in a Speed-Vac (Rappsilber et al., >>2003<<)." . _:b546878459 . . _:b546878436 . _:b546878458 . _:b546878446 . _:b546878641 . _:b546878420 . _:b546878431 . . . . _:b546878675 . . . . . . . . _:b546878398 . . _:b546878354 . . _:b546878317 . . _:b29436395 . . . . . _:b546878367 . _:b546878653 . _:b546878450 . . . . _:b546878381 . . _:b546878587 . . . _:b29436405 "Recent studies have also demonstrated that treatment outcome depends on the tumor microenvironment (vascularization, oxygenation, recruited normal cells, etc; Chauhan et al., 2011; Jacobetz et al., >>2013<<)." . . _:b546878638 . _:b29436423 "The poorly metastatic MDA-MB-231 cell line was established from cells isolated from a pleural effusion sample from a triple-negative breast cancer patient (Cailleau et al., >>1978<<). The highly metastatic MDA-MB-231-LM2 line (denoted LM2), was previously selected and characterized for increased metastatic potential to the lungs (Minn et al., 2005). 6.5 weeks post-injection, the primary tumors were harvested, ECM" . "PMC0" . . _:b546878610 . . . . . . _:b546878707 . . . . _:b546878733 . . _:b546878329 . . _:b29436450 . _:b546878429 . . _:b546878314 . _:b29436409 "(Hynes, >>2009<<). Pathologists have used excessive ECM deposition (desmoplasia) as a marker of tumors with poor prognosis long before the complexity of the ECM was even deciphered (Anastassiades and Pryce, 1974)." . . _:b546878396 . _:b546878567 . . _:b29436432 . . . _:b546878402 . _:b546878619 . _:b546878665 . _:b546878575 . _:b29436418 . _:b29436431 "Examples of these are angiopoietin-like 4 (ANGPTL4, Padua et al., 2008), cathepsin B (CTSB, Sevenich et al., 2010, 2011; Vasiljeva et al., >>2006<<), insulin-like growth factor-binding protein 4 (IGFBP4, Ryan et al., 2009) and lysyl-oxidase-like 2 (LOXL2, Barry-Hamilton et al., 2010)." . _:b546878600 . _:b546878666 . . _:b546878298 . . _:b546878451 . _:b546878460 . _:b29436420 . _:b546878659 . _:b546878476 . _:b546878736 . _:b29436458 "proteins and their cellular receptors (Goodman and Picard, 2012) or by disrupting the architecture of the ECM (that acts as a barrier) to allow more efficient drug delivery (Yuan et al., 2007; Whatcott et al., 2011; Jacobetz et al., >>2013<<), the proteins discovered in our differential proteomics-based approach have the potential to serve as novel anticancer therapeutic targets." . _:b546878720 . . . _:b546878598 . . . . _:b546878734 . . . _:b546878385 . . _:b546878738 . _:b546878371 . . . . _:b546878742 . _:b546878323 . _:b546878723 . _:b546878453 . _:b546878523 . . . . _:b546878593 . _:b546878318 . . _:b546878597 . . _:b546878701 . _:b546878335 . _:b546878430 . _:b546878373 . . _:b546878470 . _:b546878683 . . _:b546878555 . _:b29436429 . _:b546878513 . _:b546878544 . . . _:b546878652 . _:b29436427 "In fact, we have previously reported the expression of these proteins in several normal tissues (Naba et al., >>2012<<), which suggests that these proteins are widely expressed." . _:b546878473 . . _:b29436453 . _:b546878389 . _:b546878718 . . . . _:b29436393 "introduction" . . . . . . . _:b29436450 "belongs to the S100 family of acute-phase response factors and, when overexpressed in non-small-cell lung carcinoma cells subsequently injected subcutaneously in mice, has been shown to promote metastasis to the lungs (Bulk et al., >>2009<<). The fact that we observed a decrease in the number of metastases formed by cells in which S100A2 was knockeddown, both when the cells were injected orthotopically and metastasized from the primary tumor and when the cells were injected" . _:b546878645 . _:b546878358 . _:b546878647 . _:b546878347 . . _:b546878425 . _:b546878479 . . . _:b546878702 . _:b29436430 . _:b546878611 . . _:b29436457 . . _:b546878437 . _:b546878589 . _:b546878410 . _:b546878469 . _:b546878468 . . _:b546878471 . _:b546878428 . . _:b546878455 . . . _:b546878470 . . _:b546878545 . _:b546878651 . _:b546878465 . _:b546878721 . . _:b546878464 . . _:b546878539 . _:b546878636 . _:b546878467 . _:b29436443 "Consistent with the role of TGF\u03B2 in cancer progression (Padua and Massagu\u00E9, >>2009<<; Massague, 2012), we show that LTBP3, a protein that regulates TGF\u03B2 secretion and bioavailability (Chen et al., 2002; Munger and Sheppard, 2011) promotes primary tumor invasion and metastasis." . _:b546878466 . _:b546878477 . _:b29436407 . _:b546878483 . _:b546878476 . _:b546878300 . _:b546878386 . . _:b29436445 "LTBP3 is also a protein that contributes to the formation of the fibrillar ECM network (Ramirez and Rifkin, >>2009<<; Todorovic and Rifkin, 2012)." . _:b546878479 . . _:b546878478 . _:b546878487 . _:b29436408 . _:b546878473 . _:b546878689 . . _:b546878556 . _:b546878472 . _:b546878532 . _:b546878379 . _:b546878475 . . . _:b546878474 . . _:b546878485 . . _:b546878484 . _:b546878592 . _:b546878534 . _:b546878487 . _:b546878478 . _:b546878486 . _:b546878320 . _:b546878481 . _:b546878319 . . _:b546878480 . _:b546878700 . _:b546878422 . . _:b546878483 . . _:b546878482 . _:b546878493 . _:b29436454 "also serve as anchors to deliver selectively to tumors and/or metastases imaging probes to enhance the detection of metastases or anti-tumor agents to enhance therapy on the model of the work pioneered by Neri et al. (Pasche and Neri, >>2012<<). Finally, another possible translational aspect is the identification of potential novel direct therapeutic targets." . _:b546878492 . _:b546878631 . _:b546878583 . . _:b546878495 . . . _:b546878494 . _:b546878648 . . _:b546878489 . _:b546878488 . . _:b546878491 . . _:b546878489 . _:b546878490 . . _:b546878643 . _:b546878501 . _:b29436442 "Previous studies had reported the up-regulation of CYR61 in more aggressive tumor cell lines or lesions (Tsai et al., 2000, >>2002<<). Our study demonstrates that although produced in greater abundance by highly metastatic tumors, CYR61 is not necessary to promote metastatic dissemination as knocking down its expression did not have any effect on the metastatic" . _:b546878500 . _:b546878503 . . _:b546878502 . _:b546878481 . _:b546878497 . _:b546878708 . . _:b29436456 "by targeting the interactions between ECM proteins and their cellular receptors (Goodman and Picard, 2012) or by disrupting the architecture of the ECM (that acts as a barrier) to allow more efficient drug delivery (Yuan et al., >>2007<<; Whatcott et al., 2011; Jacobetz et al., 2013), the proteins discovered in our differential proteomics-based approach have the potential to serve as novel anticancer therapeutic targets." . . _:b546878496 . _:b546878499 . . _:b29436452 "aspect of tumor\u2013stroma interactions has not been extensively studied in vivo, although a recent paper notes that cancer-associated fibroblasts from different mammary tumor subtypes differ in their expression profiles (Tchou et al., >>2012<<)." . _:b546878667 . _:b546878321 . _:b546878498 . . . _:b546878509 . . . . _:b546878508 . _:b546878608 . _:b546878625 . . _:b546878511 . _:b546878510 . _:b546878505 . _:b546878504 . _:b546878518 . _:b546878507 . _:b546878309 . _:b546878670 . _:b546878506 . _:b546878584 . _:b546878378 . _:b546878517 . _:b546878686 . . . _:b29436411 "Despite its great importance in physiological (development, aging) and pathological processes such as cancer, the extracellular matrix remains underexplored (Wilson, >>2010<<)." . _:b546878516 . _:b546878519 . _:b546878377 . _:b546878435 . _:b546878518 . _:b29436399 . _:b546878725 . _:b546878513 . _:b546878668 . _:b546878527 . . _:b546878512 . . _:b546878515 . _:b546878514 . _:b546878525 . _:b29436421 "com/), an online-available meta-analysis tool (Gy\u00F6rffy et al., >>2010<<), was used to test possible correlations between expression of the genes encoding proteins identified by proteomics in our study (\u2018Results\u2019) with distant metastasis-free survival." . _:b546878303 . _:b546878524 . _:b546878527 . _:b546878599 . _:b546878526 . . . _:b546878521 . _:b546878394 . _:b29436435 "Interestingly, a recent publication by Curran and Keely highlights the convergence of TGF\u03B2 and HIF1\u03B1 signaling pathways as a key contributor to the cross-talk between breast tumor cells and stromal cells (Curran and Keely, >>2013<<). Altogether, this analysis indicates that by interrogating the tumor extracellular matrix, one can identify signaling pathways up-regulated within the tumor cells or stromal cells and these represent potential targets for therapeutic" . _:b546878520 . _:b546878523 . _:b546878522 . _:b546878684 . _:b546878533 . . . _:b546878532 . . _:b29436401 "cross-talk and interaction of the tumor cells with their surrounding microenvironment is necessary for tumor progression (Tlsty and Coussens, 2006; Polyak et al., 2009; Dvorak et al., 2011; Boudreau et al., 2012; Conklin and Keely, >>2012<<; Fordyce et al., 2012)." . _:b29436409 . _:b546878535 . _:b546878534 . _:b546878515 . _:b546878529 . . _:b546878528 . _:b546878382 . _:b546878521 . . _:b546878531 . _:b546878439 . . . _:b29436428 . _:b546878530 . _:b546878395 . _:b546878520 . _:b546878337 . _:b29436451 "In addition, analyses by Kaplan\u2013Meier Plotter (Gy\u00F6rffy et al., >>2010<<) show that several proteins from our list of ECM proteins up-regulated in highly metastatic LM2 cells (ADAM9, LOXL2, S100A2; data not shown) show statistically significant correlations with poor prognosis for metastasis-free survival of" . _:b546878541 . . _:b546878572 . _:b546878540 . . . _:b546878543 . . _:b546878469 "2"^^ . _:b546878408 . _:b546878542 . . _:b546878679 . _:b546878531 . _:b546878468 "2"^^ . _:b546878537 . . _:b546878471 "2"^^ . _:b546878536 . _:b546878331 . _:b546878470 "2"^^ . _:b29436422 "results" . _:b546878539 . _:b546878465 "2"^^ . _:b546878601 . _:b546878360 . _:b546878538 . . . _:b546878464 "2"^^ . _:b546878549 . _:b546878467 "2"^^ . _:b546878548 . . _:b546878466 "2"^^ . _:b29436405 . . _:b546878551 . . . _:b546878477 "2"^^ . _:b546878550 . . _:b546878476 "2"^^ . . _:b546878545 . . . _:b546878479 "2"^^ . _:b546878544 . _:b546878478 "2"^^ . _:b546878540 . _:b546878547 . . . _:b546878473 "2"^^ . _:b546878546 . _:b546878344 . _:b546878472 "2"^^ . _:b546878557 . _:b546878342 . . _:b546878423 . _:b546878327 . _:b546878475 "2"^^ . . _:b546878556 . _:b546878680 . _:b546878739 . _:b546878474 "2"^^ . _:b546878559 . . . _:b546878519 . _:b546878485 "2"^^ . _:b546878558 . . _:b546878484 "2"^^ . . _:b546878553 . . _:b546878487 "2"^^ . . _:b29436423 . _:b546878552 . . . _:b546878486 "2"^^ . _:b546878555 . _:b546878481 "2"^^ . _:b546878554 . . _:b546878480 "2"^^ . . _:b546878565 . _:b546878483 "2"^^ . _:b546878564 . _:b546878626 . _:b546878650 . _:b546878482 "2"^^ . _:b29436448 . _:b546878567 . . . _:b546878493 "2"^^ . _:b546878566 . . _:b546878492 "2"^^ . _:b546878561 . _:b546878591 . . _:b546878588 . _:b546878495 "2"^^ . . _:b546878560 . . _:b546878727 . _:b546878695 . . _:b546878494 "2"^^ . _:b546878326 . _:b546878463 . _:b546878563 . _:b546878464 . _:b546878489 "2"^^ . _:b546878562 . _:b546878412 . _:b546878488 "2"^^ . _:b29436404 . _:b546878573 . _:b546878449 . . . _:b546878491 "2"^^ . _:b546878388 . _:b546878572 . . . _:b546878490 "2"^^ . _:b546878575 . _:b546878397 . _:b546878501 "2"^^ . _:b546878574 . _:b546878500 "2"^^ . _:b546878569 . _:b546878503 "2"^^ . _:b546878568 . _:b29436439 . . _:b546878502 "2"^^ . _:b546878571 . . _:b546878497 "2"^^ . _:b546878570 . _:b29436449 "Klotsche-van Ameln et al. have reported that inhibiting EGLN1 had an anti-tumor effect by enhancing TGF\u03B2 anti-proliferative effect in a model of murine osteosarcoma (Klotzsche-von Ameln et al., >>2011<<). However, the extracellular role of this protein remains unknown." . _:b546878496 "2"^^ . _:b546878581 . _:b546878713 . _:b546878499 "2"^^ . _:b546878580 . . _:b546878498 "2"^^ . _:b546878583 . _:b546878509 "2"^^ . _:b29436447 . _:b546878582 . _:b546878506 . _:b546878508 "2"^^ . _:b546878577 . _:b546878511 "2"^^ . _:b546878691 . _:b546878576 . _:b546878658 . _:b546878703 . _:b546878313 . _:b546878510 "2"^^ . _:b546878579 . _:b546878624 . _:b546878510 . _:b546878505 "2"^^ . _:b546878578 . _:b546878504 "2"^^ . . _:b546878589 . . . _:b546878507 "2"^^ . _:b546878588 . _:b546878698 . _:b546878506 "2"^^ . _:b546878591 . _:b546878517 "2"^^ . . _:b546878620 . _:b546878606 . _:b546878590 . _:b546878516 "2"^^ . _:b546878585 . _:b546878629 . _:b546878519 "2"^^ . _:b546878584 . . _:b546878518 "2"^^ . _:b546878587 . _:b546878565 . _:b546878513 "2"^^ . _:b546878586 . . _:b546878512 "2"^^ . _:b546878597 . _:b546878515 "2"^^ . _:b546878596 . . _:b546878514 "2"^^ . _:b546878599 . _:b546878324 . _:b546878525 "2"^^ . _:b546878598 . . _:b546878524 "2"^^ . _:b546878593 . _:b546878527 "2"^^ . _:b546878692 . _:b546878592 . _:b546878609 . . _:b546878526 "2"^^ . _:b546878595 . . _:b546878521 "2"^^ . _:b546878594 . _:b546878520 "2"^^ . _:b29436445 . _:b546878605 . _:b546878474 . . . _:b29436447 "SNED1 (Sushi, Nidogen, and EGF-like Domains 1) is a large ECM glycoprotein first identified as a potential component of the basement membrane (Leimeister et al., >>2004<<) whose roles in normal physiology and pathology remain to be characterized." . _:b546878523 "2"^^ . _:b546878569 . _:b546878604 . _:b546878375 . _:b546878522 "2"^^ . _:b546878607 . _:b546878391 . _:b546878709 . _:b546878494 . . _:b546878533 "2"^^ . _:b29436434 "Interestingly, the LM2 cell line and its metastatic potential have previously been shown to be dependent on TGF\u03B2 (Minn et al., >>2007<<).10.7554/eLife.01308.010Figure 4.The TGF\u03B2 and the HIF1\u03B1/VEGF pathways are up-regulated in highly metastatic mammary tumors." . _:b546878606 . _:b546878548 . . _:b29436452 . _:b546878532 "2"^^ . _:b546878601 . _:b546878705 . _:b546878535 "2"^^ . _:b29436444 . _:b546878600 . _:b546878534 "2"^^ . . _:b29436446 "LTBP3 is also a protein that contributes to the formation of the fibrillar ECM network (Ramirez and Rifkin, 2009; Todorovic and Rifkin, >>2012<<). Thus, whether LTBP3 contributes to tumor progression because of its signaling role or its architectural role remains to be elucidated. Of note, LTBP3 is a member of a family of proteins and the three other members of the family; LTBP1," . _:b546878603 . _:b546878529 "2"^^ . . _:b546878602 . . _:b546878581 . . _:b546878528 "2"^^ . . _:b546878613 . . _:b29436440 "discussion" . _:b546878531 "2"^^ . . _:b546878612 . _:b546878530 "2"^^ . . _:b546878615 . _:b546878580 . . _:b546878740 . _:b546878541 "2"^^ . _:b546878496 . . _:b546878614 . _:b546878586 . _:b546878671 . _:b546878540 "2"^^ . _:b546878609 . _:b546878471 . _:b546878543 "2"^^ . _:b546878608 . _:b29436442 . _:b546878542 "2"^^ . . _:b546878611 . _:b546878674 . _:b546878537 "2"^^ . _:b29436408 "Cells are in turn able to adhere to the extracellular matrix via different types of receptors including the integrins (Hynes, 2002; Geiger and Yamada, >>2011<<). In addition to providing biophysical cues, the ECM provides biochemical signals that are major regulators of cell proliferation, survival, migration, and invasion. (Hynes, 2009). Pathologists have used excessive ECM deposition" . . _:b546878610 . _:b546878724 . . _:b546878536 "2"^^ . . _:b546878621 . . _:b546878539 "2"^^ . _:b29436436 . . _:b546878620 . _:b546878366 . . _:b546878538 "2"^^ . _:b546878623 . . _:b546878549 "2"^^ . _:b546878622 . . _:b546878548 "2"^^ . . _:b546878617 . _:b29436404 "In addition, the nature of the tumor microenvironment or gene expression profile of the stromal cells has been used to define human breast cancer types (Bergamaschi et al., 2008; Finak et al., 2008; Conklin et al., >>2011<<). Recent studies have also demonstrated that treatment outcome depends on the tumor microenvironment (vascularization, oxygenation, recruited normal cells, etc; Chauhan et al., 2011; Jacobetz et al., 2013)." . _:b546878551 "2"^^ . _:b546878616 . _:b546878550 "2"^^ . _:b546878619 . . . _:b546878545 "2"^^ . _:b546878618 . _:b546878315 . _:b546878339 . _:b29436394 . _:b546878544 "2"^^ . . _:b546878629 . _:b546878495 . _:b546878404 . _:b546878547 "2"^^ . _:b546878628 . . _:b546878546 "2"^^ . . _:b546878631 . _:b546878498 . _:b546878557 "2"^^ . _:b546878630 . _:b546878556 "2"^^ . . _:b546878625 . . . _:b546878564 . _:b546878559 "2"^^ . _:b546878475 . _:b546878624 . . _:b546878558 "2"^^ . _:b546878627 . _:b546878574 . _:b546878553 "2"^^ . _:b546878626 . _:b546878552 "2"^^ . _:b546878637 . _:b546878393 . _:b546878555 "2"^^ . . . _:b546878636 . . . _:b546878554 "2"^^ . _:b546878639 . _:b546878406 . _:b546878565 "2"^^ . _:b546878638 . _:b546878564 "2"^^ . . _:b546878633 . _:b546878577 . . _:b546878567 "2"^^ . _:b546878424 . _:b546878632 . _:b29436441 "Previous studies had reported the up-regulation of CYR61 in more aggressive tumor cell lines or lesions (Tsai et al., >>2000<<, 2002). Our study demonstrates that although produced in greater abundance by highly metastatic tumors, CYR61 is not necessary to promote metastatic dissemination as knocking down its expression did not have any effect on the metastatic" . _:b546878566 "2"^^ . _:b29436454 . _:b546878635 . . _:b546878543 . _:b546878561 "2"^^ . _:b546878634 . _:b546878634 . . _:b546878560 "2"^^ . _:b546878645 . . . _:b546878563 "2"^^ . . _:b546878644 . . _:b546878562 "2"^^ . _:b546878647 . _:b546878505 . _:b546878415 . _:b546878573 "2"^^ . _:b546878646 . _:b546878533 . _:b546878572 "2"^^ . _:b546878362 . _:b546878641 . _:b546878380 . _:b546878575 "2"^^ . . _:b546878640 . _:b546878574 "2"^^ . _:b29436422 . _:b546878643 . _:b29436412 "and tumors, we have developed a proteomics-based method to enrich and identify ECM proteins and coupled it with a bioinformatic annotation of the \u2018matrisome\u2019 defined as the ensemble of ECM and ECM-associated proteins (Naba et al., >>2012<<). Using this approach, we characterized the extracellular matrices of normal murine tissues (e.g., lung and colon) and demonstrated that each of these comprises over 100 proteins." . . _:b546878569 "2"^^ . _:b546878642 . _:b546878541 . _:b546878568 "2"^^ . . _:b546878653 . . . _:b29436455 "Whether by targeting the interactions between ECM proteins and their cellular receptors (Goodman and Picard, >>2012<<) or by disrupting the architecture of the ECM (that acts as a barrier) to allow more efficient drug delivery (Yuan et al., 2007; Whatcott et al., 2011; Jacobetz et al., 2013), the proteins discovered in our differential proteomics-based" . _:b29436440 . . _:b546878306 . _:b29436455 . _:b546878571 "2"^^ . _:b546878652 . _:b29436420 "Retroviruses were packaged in 293FT cells and LM2 cells were transduced as previously described (Stern et al., 2008; Lamar et al., >>2012<<). For qPCR, RNA was isolated from cell or tumor lysates using RNeasy kit (Qiagen, Germantown, MD) and cDNA was synthesized by reverse transcription using the First-Strand cDNA Synthesis Kit (Promega, Madison, WI). qPCR reactions were" . . . _:b546878570 "2"^^ . _:b546878655 . . _:b546878685 . . _:b546878581 "2"^^ . _:b546878654 . _:b546878338 . _:b546878580 "2"^^ . . _:b546878649 . _:b546878307 . _:b546878583 "2"^^ . _:b546878648 . _:b29436395 "Indeed, in order to progress, a tumor needs to be surrounded by a permissive environment and, to metastasize, tumor cells need to find or create a favorable niche (seed and soil theory) (Paget, 1889; Ribatti et al., >>2006<<; Erler and Weaver, 2009; Comen, 2012)." . _:b546878657 . . _:b546878582 "2"^^ . _:b546878651 . . . . _:b546878577 "2"^^ . _:b546878650 . _:b546878409 . _:b546878468 . _:b546878576 "2"^^ . _:b546878661 . _:b546878662 . _:b546878579 "2"^^ . _:b546878660 . _:b29436425 "6.5 weeks post-injection, the primary tumors were harvested, ECM proteins were enriched from tumors using the subcellular fractionation protocol described previously (Naba et al. >>2012<<), and the composition of the ECM-enriched fractions obtained was characterized by mass spectrometry (Figure 1A)." . _:b29436417 "type=shRNA), and cloned into the MSCV-ZSG-2A-Puro-miR30 vector retroviral vector as described previously (Stern et al., >>2008<<; Lamar et al., 2012)." . . _:b546878730 . _:b546878630 . _:b546878578 "2"^^ . _:b546878663 . _:b546878589 "2"^^ . . _:b546878662 . . _:b546878588 "2"^^ . _:b546878657 . _:b29436400 . _:b546878612 . _:b546878591 "2"^^ . _:b546878656 . _:b546878590 "2"^^ . _:b546878659 . _:b546878711 . _:b546878585 "2"^^ . _:b546878658 . _:b546878584 "2"^^ . _:b546878669 . _:b546878587 "2"^^ . _:b546878687 . _:b546878668 . _:b546878384 . _:b546878586 "2"^^ . _:b546878456 . _:b546878671 . _:b546878597 "2"^^ . _:b546878670 . . _:b546878596 "2"^^ . . _:b546878665 . . _:b546878599 "2"^^ . _:b546878664 . _:b546878598 "2"^^ . _:b546878667 . _:b546878593 "2"^^ . _:b29436401 . _:b546878615 . _:b546878666 . . _:b546878704 . _:b546878592 "2"^^ . _:b546878677 . _:b29436396 "in order to progress, a tumor needs to be surrounded by a permissive environment and, to metastasize, tumor cells need to find or create a favorable niche (seed and soil theory) (Paget, 1889; Ribatti et al., 2006; Erler and Weaver, >>2009<<; Comen, 2012). To create such a niche, tumor cells either directly alter the microenvironment, or instruct local or recruited stromal cells to do so." . _:b546878333 . _:b29436403 "In addition, the nature of the tumor microenvironment or gene expression profile of the stromal cells has been used to define human breast cancer types (Bergamaschi et al., >>2008<<; Finak et al., 2008; Conklin et al., 2011)." . _:b546878595 "2"^^ . _:b546878676 . . _:b546878594 "2"^^ . . . _:b546878679 . . . _:b546878605 "2"^^ . _:b546878550 . _:b546878678 . . _:b546878604 "2"^^ . _:b29436429 "Examples of these are angiopoietin-like 4 (ANGPTL4, Padua et al., 2008), cathepsin B (CTSB, Sevenich et al., >>2010<<, 2011; Vasiljeva et al., 2006), insulin-like growth factor-binding protein 4 (IGFBP4, Ryan et al., 2009) and lysyl-oxidase-like 2 (LOXL2, Barry-Hamilton et al., 2010)." . _:b546878673 . . _:b546878573 . _:b546878607 "2"^^ . . _:b546878672 . _:b546878606 "2"^^ . _:b546878607 . . _:b546878675 . . . _:b546878601 "2"^^ . _:b546878674 . _:b29436415 "Sequential extractions of proteins from frozen tumor samples were performed using the CNMCS (Cytosol/Nucleus/Membrane/Cytoskeleton) compartmental protein extraction kit (Cytomol, Union City, CA) as previously described (Naba et al. >>2012<<). This led to the extraction of intracellular soluble proteins and the enrichment of ECM proteins." . . _:b546878600 "2"^^ . _:b546878305 . _:b546878685 . _:b546878418 . _:b546878530 . . _:b546878603 "2"^^ . . _:b546878684 . _:b546878655 . . _:b546878602 "2"^^ . _:b546878682 . _:b546878687 . _:b546878722 . _:b546878613 "2"^^ . _:b546878686 . _:b546878612 "2"^^ . _:b546878681 . . . _:b546878615 "2"^^ . _:b546878680 . _:b546878618 . _:b546878614 "2"^^ . . _:b546878683 . . _:b546878528 . . _:b546878609 "2"^^ . _:b546878682 . _:b546878441 . _:b546878608 "2"^^ . _:b546878693 . . _:b546878611 "2"^^ . _:b546878692 . . . _:b29436396 . _:b546878610 "2"^^ . _:b546878695 . . . _:b29436397 . _:b546878621 "2"^^ . _:b546878694 . . _:b546878363 . _:b29436398 . . _:b546878620 "2"^^ . _:b546878558 . _:b546878689 . . . _:b29436399 . _:b546878623 "2"^^ . . _:b546878688 . _:b29436393 _:b29436394 . _:b29436393 _:b29436395 . _:b29436393 _:b29436396 . _:b546878622 "2"^^ . _:b29436393 _:b29436397 . _:b546878691 . _:b29436393 _:b29436398 . . _:b29436393 _:b29436399 . _:b29436393 _:b29436400 . _:b546878617 "2"^^ . _:b29436393 . _:b29436393 _:b29436401 . _:b546878690 . _:b29436393 _:b29436402 . _:b546878637 . _:b29436393 _:b29436403 . _:b29436394 . _:b29436393 _:b29436404 . _:b546878616 "2"^^ . _:b29436393 _:b29436405 . _:b546878701 . _:b546878524 . _:b29436393 _:b29436406 . . _:b29436393 _:b29436407 . _:b29436395 . _:b546878616 . . _:b546878619 "2"^^ . _:b29436393 _:b29436408 . _:b29436393 _:b29436409 . _:b546878700 . _:b29436393 _:b29436410 . _:b29436393 _:b29436411 . _:b29436404 . _:b546878664 . _:b29436393 _:b29436412 . _:b546878618 "2"^^ . _:b546878703 . _:b29436405 . _:b546878629 "2"^^ . _:b546878702 . . _:b29436406 . _:b546878628 "2"^^ . _:b546878697 . _:b546878454 . . _:b29436407 . . _:b546878631 "2"^^ . _:b546878546 . _:b546878696 . _:b29436400 . . _:b546878630 "2"^^ . _:b546878699 . _:b546878458 . _:b29436401 . _:b546878625 "2"^^ . _:b546878698 . _:b29436402 . _:b546878624 "2"^^ . _:b546878709 . . _:b29436403 . _:b546878627 "2"^^ . _:b546878708 . _:b546878480 . _:b29436412 . _:b546878626 "2"^^ . _:b546878711 . _:b29436413 . _:b546878637 "2"^^ . _:b546878710 . _:b546878716 . _:b29436414 . . _:b546878636 "2"^^ . _:b546878509 . _:b546878705 . _:b546878387 . _:b29436434 . _:b29436415 . _:b546878639 "2"^^ . _:b546878704 . _:b29436408 . _:b546878638 "2"^^ . _:b546878707 . _:b29436413 _:b29436414 . _:b29436413 _:b29436415 . _:b29436409 . . _:b546878633 "2"^^ . . _:b546878706 . _:b546878359 . _:b29436410 . _:b546878632 "2"^^ . _:b546878717 . . _:b29436411 . _:b29436397 "progress, a tumor needs to be surrounded by a permissive environment and, to metastasize, tumor cells need to find or create a favorable niche (seed and soil theory) (Paget, 1889; Ribatti et al., 2006; Erler and Weaver, 2009; Comen, >>2012<<). To create such a niche, tumor cells either directly alter the microenvironment, or instruct local or recruited stromal cells to do so." . _:b546878635 "2"^^ . . _:b546878716 . . _:b546878634 "2"^^ . . _:b546878719 . _:b546878714 . _:b546878312 . _:b546878645 "2"^^ . . _:b546878718 . _:b546878644 "2"^^ . . _:b546878713 . _:b546878502 . . _:b546878647 "2"^^ . . _:b546878712 . _:b546878646 "2"^^ . _:b546878715 . . . _:b546878559 . _:b546878641 "2"^^ . _:b546878714 . _:b546878399 . _:b546878562 . _:b546878640 "2"^^ . _:b546878643 "2"^^ . _:b546878694 . _:b546878642 "2"^^ . _:b546878308 . _:b546878653 "2"^^ . _:b546878652 "2"^^ . _:b546878334 . _:b546878655 "2"^^ . . _:b546878514 . _:b546878654 "2"^^ . _:b546878649 "2"^^ . _:b546878613 . _:b546878648 "2"^^ . . . _:b546878651 "2"^^ . . . _:b546878650 "2"^^ . . _:b546878661 "2"^^ . . _:b546878660 "2"^^ . . . _:b546878663 "2"^^ . . _:b546878557 . _:b546878403 . _:b546878662 "2"^^ . . . . _:b546878657 "2"^^ . _:b546878656 "2"^^ . _:b546878659 "2"^^ . . _:b546878603 . _:b546878658 "2"^^ . _:b546878485 . _:b546878549 . _:b29436402 . _:b546878669 "2"^^ . . . _:b546878668 "2"^^ . . . _:b546878568 . _:b546878671 "2"^^ . . _:b546878501 . _:b546878407 . _:b546878670 "2"^^ . _:b546878511 . . _:b546878665 "2"^^ . _:b546878372 . _:b29436424 "The highly metastatic MDA-MB-231-LM2 line (denoted LM2), was previously selected and characterized for increased metastatic potential to the lungs (Minn et al., >>2005<<). 6.5 weeks post-injection, the primary tumors were harvested, ECM proteins were enriched from tumors using the subcellular fractionation protocol described previously (Naba et al. 2012), and the composition of the ECM-enriched fractions" . _:b546878656 . . _:b546878664 "2"^^ . . _:b546878667 "2"^^ . _:b546878374 . _:b546878666 "2"^^ . . . _:b546878677 "2"^^ . _:b29436443 . . _:b546878676 "2"^^ . . . . _:b546878679 "2"^^ . . _:b546878678 "2"^^ . . _:b546878353 . _:b29436419 "Retroviruses were packaged in 293FT cells and LM2 cells were transduced as previously described (Stern et al., >>2008<<; Lamar et al., 2012)." . . _:b546878673 "2"^^ . _:b29436426 . _:b546878719 . _:b546878635 . _:b546878646 . _:b546878672 "2"^^ . _:b29436427 . _:b29436438 "We used Kaplan\u2013Meier Plotter (Gy\u00F6rffy et al., >>2010<<), an online-available meta-analysis tool for biomarker assessment." . _:b29436456 . _:b546878477 . _:b546878675 "2"^^ . _:b546878512 . . . _:b546878674 "2"^^ . _:b29436425 . _:b546878685 "2"^^ . _:b546878571 . _:b546878684 "2"^^ . _:b546878687 "2"^^ . . _:b546878332 . _:b546878333 . _:b546878334 . _:b546878686 "2"^^ . . _:b546878335 . _:b546878328 . _:b546878329 . _:b546878330 . _:b29436417 . _:b546878681 "2"^^ . _:b546878331 . _:b546878324 . _:b546878462 . _:b546878459 . _:b546878325 . _:b546878552 . _:b546878326 . _:b546878680 "2"^^ . . . _:b546878327 . _:b546878320 . _:b546878321 . _:b546878322 . _:b29436419 . _:b546878683 "2"^^ . _:b546878323 . . _:b546878316 . _:b546878317 . _:b546878318 . _:b546878682 "2"^^ . _:b546878319 . _:b546878312 . _:b546878313 . _:b546878314 . _:b546878693 "2"^^ . . _:b546878315 . _:b546878308 . _:b546878309 . _:b546878390 . _:b546878692 "2"^^ . _:b546878310 . _:b546878311 . _:b546878304 . _:b546878305 . . _:b546878695 "2"^^ . _:b546878306 . . _:b546878307 . _:b546878300 . _:b546878301 . . _:b546878694 "2"^^ . _:b546878499 . _:b546878302 . _:b546878303 . . _:b546878297 . _:b546878298 . _:b546878689 "2"^^ . _:b546878299 . _:b546878457 . _:b546878688 "2"^^ . _:b546878432 . _:b546878691 "2"^^ . _:b546878416 . _:b546878681 . _:b546878690 "2"^^ . _:b29436415 . . _:b546878701 "2"^^ . _:b546878369 . _:b29436412 . _:b546878700 "2"^^ . _:b29436406 "The extracellular matrix (ECM) is the complex scaffold of proteins that provides the architectural support for cell and tissue organization (Hynes and Naba, >>2012<<). Cells are in turn able to adhere to the extracellular matrix via different types of receptors including the integrins (Hynes, 2002; Geiger and Yamada, 2011)." . . _:b546878703 "2"^^ . . _:b546878396 . _:b546878397 . _:b546878398 . _:b546878702 "2"^^ . _:b546878399 . _:b546878392 . _:b546878393 . _:b546878394 . _:b29436398 . _:b546878697 "2"^^ . _:b546878395 . _:b546878731 . . . _:b546878343 . _:b546878388 . _:b546878389 . _:b546878390 . _:b546878696 "2"^^ . _:b546878461 . _:b546878341 . _:b546878391 . _:b546878384 . _:b29436403 . _:b546878385 . _:b546878386 . _:b546878699 "2"^^ . . _:b546878387 . _:b546878380 . _:b546878381 . _:b546878382 . _:b29436433 . _:b546878698 "2"^^ . _:b546878383 . _:b546878376 . _:b546878710 . _:b546878377 . _:b546878378 . _:b546878709 "2"^^ . _:b546878379 . _:b546878372 . _:b546878373 . _:b546878374 . _:b546878708 "2"^^ . _:b546878375 . _:b546878368 . _:b546878303 "11"^^ . _:b546878310 . _:b546878369 . _:b29436393 . _:b546878711 "2"^^ . _:b546878370 . . _:b546878371 . . . _:b546878302 "11"^^ . _:b546878364 . . _:b546878365 . _:b546878366 . _:b546878710 "2"^^ . _:b546878297 "17"^^ . _:b546878367 . _:b546878298 "14"^^ . _:b546878360 . _:b546878553 . _:b546878301 "11"^^ . . _:b546878361 . _:b546878362 . _:b546878705 "2"^^ . _:b546878363 . _:b546878356 . _:b546878300 "11"^^ . _:b546878357 . _:b29436413 . _:b546878704 "2"^^ . _:b546878358 . _:b546878359 . _:b546878352 . _:b546878353 . . _:b546878707 "2"^^ . _:b546878299 "13"^^ . _:b546878354 . _:b546878355 . _:b546878348 . _:b546878349 . _:b546878350 . _:b546878706 "2"^^ . _:b546878351 . _:b29436402 "of the tumor cells with their surrounding microenvironment is necessary for tumor progression (Tlsty and Coussens, 2006; Polyak et al., 2009; Dvorak et al., 2011; Boudreau et al., 2012; Conklin and Keely, 2012; Fordyce et al., >>2012<<). In addition, the nature of the tumor microenvironment or gene expression profile of the stromal cells has been used to define human breast cancer types (Bergamaschi et al., 2008; Finak et al., 2008; Conklin et al., 2011)." . _:b546878344 . _:b546878345 . . _:b546878717 "2"^^ . _:b546878346 . _:b546878347 . _:b546878340 . _:b546878341 . _:b546878342 . _:b546878716 "2"^^ . _:b546878343 . _:b546878310 "6"^^ . _:b546878336 . _:b546878337 . _:b546878338 . _:b546878719 "2"^^ . . _:b546878339 . _:b546878311 "6"^^ . _:b546878460 . _:b546878461 . _:b546878462 . _:b546878718 "2"^^ . . _:b546878463 . _:b546878456 . _:b546878308 "7"^^ . . _:b546878457 . _:b546878458 . _:b546878713 "2"^^ . _:b546878459 . _:b546878699 . _:b546878452 . _:b546878309 "7"^^ . _:b546878453 . _:b546878454 . _:b546878712 "2"^^ . _:b546878455 . _:b546878448 . _:b546878449 . _:b546878306 "8"^^ . _:b546878450 . _:b546878715 "2"^^ . . _:b546878451 . _:b546878444 . _:b546878307 "7"^^ . _:b546878445 . _:b546878446 . _:b546878714 "2"^^ . _:b546878447 . _:b546878440 . _:b546878441 . _:b546878442 . _:b546878725 "2"^^ . _:b546878443 . _:b546878304 "10"^^ . _:b546878436 . _:b546878639 . . _:b546878437 . . _:b546878438 . _:b546878724 "2"^^ . _:b546878305 "9"^^ . _:b546878439 . _:b546878432 . . _:b546878433 . _:b546878434 . _:b546878727 "2"^^ . _:b546878482 . _:b546878317 "5"^^ . _:b546878435 . . _:b546878428 . _:b546878429 . _:b546878430 . _:b546878726 "2"^^ . _:b546878316 "5"^^ . _:b546878431 . _:b546878424 . _:b546878425 . _:b546878426 . _:b546878721 "2"^^ . _:b546878319 "5"^^ . _:b546878427 . _:b546878420 . . _:b546878421 . _:b546878422 . _:b546878720 "2"^^ . _:b546878318 "5"^^ . _:b546878423 . _:b546878314 "6"^^ . _:b546878401 . _:b546878416 . . _:b546878448 . _:b546878417 . _:b546878418 . _:b546878723 "2"^^ . _:b546878419 . _:b546878315 "6"^^ . _:b546878412 . _:b546878413 . _:b546878414 . _:b546878722 "2"^^ . _:b546878415 . _:b546878312 "6"^^ . _:b546878408 . _:b546878576 . _:b546878438 . _:b546878409 . _:b546878410 . _:b29436444 "Consistent with the role of TGF\u03B2 in cancer progression (Padua and Massagu\u00E9, 2009; Massague, 2012), we show that LTBP3, a protein that regulates TGF\u03B2 secretion and bioavailability (Chen et al., >>2002<<; Munger and Sheppard, 2011) promotes primary tumor invasion and metastasis. LTBP3 is also a protein that contributes to the formation of the fibrillar ECM network (Ramirez and Rifkin, 2009; Todorovic and Rifkin, 2012)." . _:b546878733 "2"^^ . _:b546878411 . _:b546878313 "6"^^ . _:b546878404 . _:b546878405 . _:b546878602 . _:b546878406 . _:b546878732 "2"^^ . _:b546878407 . _:b546878400 . _:b546878401 . _:b546878726 . . _:b546878402 . _:b29436407 "Cells are in turn able to adhere to the extracellular matrix via different types of receptors including the integrins (Hynes, >>2002<<; Geiger and Yamada, 2011)." . _:b546878735 "2"^^ . _:b546878325 "5"^^ . _:b546878403 . _:b546878524 . _:b546878525 . _:b546878526 . _:b546878734 "2"^^ . _:b546878324 "5"^^ . _:b546878527 . . _:b546878520 . _:b29436458 . _:b546878521 . _:b546878522 . _:b546878729 "2"^^ . _:b546878443 . . _:b546878327 "5"^^ . _:b546878523 . . _:b546878516 . _:b546878417 . _:b546878517 . _:b29436438 . . . _:b546878728 "2"^^ . _:b546878326 "5"^^ . _:b546878518 . _:b546878519 . _:b546878301 . _:b546878512 . _:b546878513 . . _:b546878514 . _:b546878731 "2"^^ . _:b546878321 "5"^^ . _:b546878515 . _:b546878628 . _:b546878300 . _:b546878508 . _:b29436453 "Development of immuno- or mass spectrometry-based assays (Gillette and Carr, >>2013<<) for these proteins is therefore of interest since protein-level measurements may be of equivalent or greater predictive value than RNA expression analyses." . . _:b546878509 . . _:b546878510 . _:b546878730 "2"^^ . _:b546878320 "5"^^ . _:b546878511 . _:b546878303 . _:b546878504 . _:b546878505 . _:b546878506 . _:b546878741 "2"^^ . _:b546878323 "5"^^ . _:b546878507 . _:b546878302 . _:b546878500 . _:b546878501 . _:b546878502 . _:b546878740 "2"^^ . _:b546878322 "5"^^ . _:b546878503 . _:b546878297 . _:b546878496 . _:b29436411 . _:b546878497 . _:b546878728 . _:b546878333 "4"^^ . _:b546878498 . _:b546878499 . . _:b546878469 . _:b546878492 . _:b546878493 . _:b546878332 "4"^^ . _:b546878494 . _:b546878742 "2"^^ . _:b546878495 . _:b546878299 . _:b546878488 . . _:b546878489 . _:b546878335 "4"^^ . _:b546878490 . _:b546878737 "2"^^ . _:b546878491 . _:b546878298 . _:b546878484 . _:b546878566 . _:b546878485 . _:b546878334 "4"^^ . _:b546878486 . _:b546878736 "2"^^ . _:b546878311 . . _:b546878487 . _:b546878309 . _:b546878480 . _:b29436420 . _:b546878481 . _:b546878482 . _:b546878739 "2"^^ . _:b546878329 "5"^^ . _:b546878483 . _:b546878308 . _:b546878476 . _:b29436421 . _:b546878477 . _:b546878478 . _:b546878738 "2"^^ . _:b546878328 "5"^^ . _:b546878479 . _:b546878311 . . _:b546878472 . . _:b29436422 . _:b546878331 "4"^^ . _:b546878473 . _:b546878474 . _:b546878475 . _:b546878310 . _:b546878468 . _:b29436423 . _:b546878469 . _:b546878470 . _:b546878330 "5"^^ . _:b546878471 . _:b546878305 . _:b546878464 . _:b29436416 . _:b546878465 . _:b546878341 "4"^^ . _:b546878466 . _:b546878467 . _:b546878304 . _:b546878588 . _:b29436417 . _:b546878589 . _:b546878340 "4"^^ . _:b546878590 . _:b546878591 . _:b546878307 . _:b546878584 . _:b29436418 . _:b546878585 . _:b546878343 "4"^^ . . _:b546878725 . _:b546878561 . _:b546878586 . _:b546878587 . _:b546878306 . _:b546878580 . _:b29436419 . _:b546878581 . _:b546878342 "4"^^ . _:b546878582 . _:b546878724 . _:b546878583 . _:b546878317 . _:b546878576 . _:b29436428 . _:b546878355 . _:b29436421 . _:b546878337 "4"^^ . _:b546878577 . _:b546878578 . _:b546878727 . _:b546878579 . _:b546878316 . _:b546878572 . _:b29436429 . _:b546878573 . _:b546878336 "4"^^ . _:b546878574 . _:b29436410 . _:b546878726 . _:b546878575 . _:b546878319 . _:b546878568 . _:b29436430 . _:b546878569 . _:b546878339 "4"^^ . . _:b546878721 . _:b546878570 . _:b546878571 . _:b29436439 "In this context, it is significant that the MDA-MB-231 cells were isolated from the pleural effusion of a triple-negative breast cancer patient (Cailleau et al., >>1978<<). While we are not suggesting that the correlations between the expression of two ECM proteins and probability of metastasis in a limited subset of cancer patients is currently of predictive value, this analysis demonstrates that we can" . _:b546878318 . _:b546878564 . _:b29436431 . . _:b546878565 . _:b546878338 "4"^^ . _:b546878566 . _:b546878720 . _:b546878567 . _:b546878313 . _:b546878560 . _:b29436424 . _:b546878349 "3"^^ . _:b546878561 . _:b546878562 . _:b546878723 . _:b546878312 . _:b546878563 . _:b546878426 . _:b546878556 . _:b29436425 . _:b546878348 "3"^^ . _:b546878557 . _:b546878558 . _:b546878722 . _:b546878315 . _:b546878559 . . _:b29436426 . _:b29436397 . _:b546878433 . _:b546878351 "3"^^ . _:b546878552 . _:b546878553 . _:b546878440 . _:b546878554 . _:b546878733 . _:b546878314 . _:b546878555 . _:b546878548 . _:b29436427 . . _:b546878350 "3"^^ . _:b546878549 . _:b29436424 . _:b546878550 . _:b29436414 . _:b546878732 . _:b546878325 . _:b546878551 . _:b546878340 . _:b29436436 . _:b546878544 . _:b546878545 . _:b546878345 "4"^^ . _:b546878546 . _:b546878735 . _:b546878324 . _:b546878547 .