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• W2011953153 abstract "Breast cancer metastasis is the leading cause of cancer-related deaths in women worldwide. Collagen in the tumor microenvironment plays a crucial role in regulating tumor progression. We have shown that type III collagen (Col3), a component of tumor stroma, regulates myofibroblast differentiation and scar formation after cutaneous injury. During the course of these wound-healing studies, we noted that tumors developed at a higher frequency in Col3+/− mice compared to wild-type littermate controls. We, therefore, examined the effect of Col3 deficiency on tumor behavior, using the murine mammary carcinoma cell line 4T1. Notably, tumor volume and pulmonary metastatic burden after orthotopic injection of 4T1 cells were increased in Col3+/− mice compared to Col3+/+ littermates. By using murine (4T1) and human (MDA-MB-231) breast cancer cells grown in Col3-poor and Col3-enriched microenvironments in vitro, we found that several major events of the metastatic process were suppressed by Col3, including adhesion, invasion, and migration. In addition, Col3 deficiency increased proliferation and decreased apoptosis of 4T1 cells both in vitro and in primary tumors in vivo. Mechanistically, Col3 suppresses the procarcinogenic microenvironment by regulating stromal organization, including density and alignment of fibrillar collagen and myofibroblasts. We propose that Col3 plays an important role in the tumor microenvironment by suppressing metastasis-promoting characteristics of the tumor-associated stroma. Breast cancer metastasis is the leading cause of cancer-related deaths in women worldwide. Collagen in the tumor microenvironment plays a crucial role in regulating tumor progression. We have shown that type III collagen (Col3), a component of tumor stroma, regulates myofibroblast differentiation and scar formation after cutaneous injury. During the course of these wound-healing studies, we noted that tumors developed at a higher frequency in Col3+/− mice compared to wild-type littermate controls. We, therefore, examined the effect of Col3 deficiency on tumor behavior, using the murine mammary carcinoma cell line 4T1. Notably, tumor volume and pulmonary metastatic burden after orthotopic injection of 4T1 cells were increased in Col3+/− mice compared to Col3+/+ littermates. By using murine (4T1) and human (MDA-MB-231) breast cancer cells grown in Col3-poor and Col3-enriched microenvironments in vitro, we found that several major events of the metastatic process were suppressed by Col3, including adhesion, invasion, and migration. In addition, Col3 deficiency increased proliferation and decreased apoptosis of 4T1 cells both in vitro and in primary tumors in vivo. Mechanistically, Col3 suppresses the procarcinogenic microenvironment by regulating stromal organization, including density and alignment of fibrillar collagen and myofibroblasts. We propose that Col3 plays an important role in the tumor microenvironment by suppressing metastasis-promoting characteristics of the tumor-associated stroma. Breast cancer is the most frequently diagnosed cancer in women and is the leading cause of cancer-related deaths in women worldwide (World Health Organization: Latest World Cancer Statistics. 2013, Press Release Number 223, http://www.iarc.fr/en/media-centre/pr/2013/pdfs/pr223_E.pdf). In fact, >500,000 women are predicted to die this year alone. Without major changes in prevention or treatment, those numbers are anticipated to nearly double in 20 years. In most patients, death is not caused by the primary tumor, but rather by metastases. The extracellular matrix (ECM) of the tumor microenvironment plays a critical role in cancer development and progression through its ability to modulate physical, biochemical, and biomechanical cues perceived by both tumor cells and cancer-associated stromal cells.1Lu P. Weaver V.M. Werb Z. The extracellular matrix: a dynamic niche in cancer progression.J Cell Biol. 2012; 196: 395-406Crossref PubMed Scopus (2022) Google Scholar, 2Dvorak H.F. Weaver V.M. Tlsty T.D. Bergers G. Tumor microenvironment and progression.J Surg Oncol. 2011; 103: 468-474Crossref PubMed Scopus (145) Google Scholar, 3Radisky E.S. Radisky D.C. Stromal induction of breast cancer: inflammation and invasion.Rev Endocr Metab Disord. 2007; 8: 279-287Crossref PubMed Scopus (104) Google Scholar Given this critical role of the ECM and tumor stromal cells in breast cancer development and progression, it is not surprising that recent studies suggest that targeting the tumor stroma is a potential therapeutic strategy for tumor control.4Lyons T.R. O'Brien J. Borges V.F. Conklin M.W. Keely P.J. Eliceiri K.W. Marusyk A. Tan A.C. Schedin P. Postpartum mammary gland involution drives progression of ductal carcinoma in situ through collagen and COX-2.Nat Med. 2011; 17: 1109-1115Crossref PubMed Scopus (266) Google Scholar, 5Tchou J. Conejo-Garcia J. Targeting the tumor stroma as a novel treatment strategy for breast cancer: shifting from the neoplastic cell-centric to a stroma-centric paradigm.Adv Pharmacol. 2012; 65: 45-61Crossref PubMed Scopus (53) Google Scholar, 6Gilkes D.M. Chaturvedi P. Bajpai S. Wong C.C. Wei H. Pitcairn S. Hubbi M.E. Wirtz D. Semenza G.L. Collagen prolyl hydroxylases are essential for breast cancer metastasis.Cancer Res. 2013; 73: 3285-3296Crossref PubMed Scopus (207) Google Scholar However, optimization of such strategies requires a better understanding of the mechanistic role that individual stromal components play in regulating tumor cell behavior.Collagen, a major component of the ECM, is increasingly recognized to play a key role in regulating breast cancer progression. Although most research on collagen in breast cancer has focused on type I collagen (Col1), and many reports have documented a negative correlation between Col1 expression and prognosis in breast cancer patients, collagen types IV, V, VI, and XVIII have also been implicated in modulation of breast cancer cell activities and fate.7Albini A. Mirisola V. Pfeffer U. Metastasis signatures: genes regulating tumor-microenvironment interactions predict metastatic behavior.Cancer Metastasis Rev. 2008; 27: 75-83Crossref PubMed Scopus (65) Google Scholar, 8Luparello C. Aspects of collagen changes in breast cancer.J Carcinog Mutagen. 2013; S13: 007Crossref Google Scholar, 9Lourenco G.J. Cardoso-Filho C. Goncales N.S. Shinzato J.Y. Zeferino L.C. Nascimento H. Costa F.F. Gurgel M.S. Lima C.S. A high risk of occurrence of sporadic breast cancer in individuals with the 104NN polymorphism of the COL18A1 gene.Breast Cancer Res Treat. 2006; 100: 335-338Crossref PubMed Scopus (22) Google Scholar, 10Barsky S.H. Rao C.N. Grotendorst G.R. Liotta L.A. Increased content of type V collagen in desmoplasia of human breast carcinoma.Am J Pathol. 1982; 108: 276-283PubMed Google Scholar, 11Robledo T. Arriaga-Pizano L. Lopez-Perez M. Salazar E.P. Type IV collagen induces STAT5 activation in MCF7 human breast cancer cells.Matrix Biol. 2005; 24: 469-477Crossref PubMed Scopus (19) Google Scholar, 12Iyengar P. Espina V. Williams T.W. Lin Y. Berry D. Jelicks L.A. Lee H. Temple K. Graves R. Pollard J. Chopra N. Russell R.G. Sasisekharan R. Trock B.J. Lippman M. Calvert V.S. Petricoin 3rd, E.F. Liotta L. Dadachova E. Pestell R.G. Lisanti M.P. Bonaldo P. Scherer P.E. Adipocyte-derived collagen VI affects early mammary tumor progression in vivo, demonstrating a critical interaction in the tumor/stroma microenvironment.J Clin Invest. 2005; 115: 1163-1176Crossref PubMed Scopus (292) Google Scholar Increased collagen density in the tumor stroma can promote invasion and metastasis of breast cancer cells.4Lyons T.R. O'Brien J. Borges V.F. Conklin M.W. Keely P.J. Eliceiri K.W. Marusyk A. Tan A.C. Schedin P. Postpartum mammary gland involution drives progression of ductal carcinoma in situ through collagen and COX-2.Nat Med. 2011; 17: 1109-1115Crossref PubMed Scopus (266) Google Scholar, 13Kakkad S.M. Solaiyappan M. Argani P. Sukumar S. Jacobs L.K. Leibfritz D. Bhujwalla Z.M. Glunde K. Collagen I fiber density increases in lymph node positive breast cancers: pilot study.J Biomed Opt. 2012; 17: 116017Crossref PubMed Scopus (79) Google Scholar, 14Maskarinec G. Pagano I.S. Little M.A. Conroy S.M. Park S.Y. Kolonel L.N. Mammographic density as a predictor of breast cancer survival: the multiethnic cohort.Breast Cancer Res. 2013; 15: R7Crossref PubMed Scopus (43) Google Scholar, 15Provenzano P.P. Inman D.R. Eliceiri K.W. Knittel J.G. Yan L. Rueden C.T. White J.G. Keely P.J. Collagen density promotes mammary tumor initiation and progression.BMC Med. 2008; 6: 11Crossref PubMed Scopus (933) Google Scholar In addition, differences in the organization and stiffness of the tumor stroma are known to influence tumor cell responses and stromal remodeling, a key step in metastasis.15Provenzano P.P. Inman D.R. Eliceiri K.W. Knittel J.G. Yan L. Rueden C.T. White J.G. Keely P.J. Collagen density promotes mammary tumor initiation and progression.BMC Med. 2008; 6: 11Crossref PubMed Scopus (933) Google Scholar, 16Conklin M.W. Eickhoff J.C. Riching K.M. Pehlke C.A. Eliceiri K.W. Provenzano P.P. Friedl A. Keely P.J. Aligned collagen is a prognostic signature for survival in human breast carcinoma.Am J Pathol. 2011; 178: 1221-1232Abstract Full Text Full Text PDF PubMed Scopus (776) Google Scholar, 17Ajeti V. Nadiarnykh O. Ponik S.M. Keely P.J. Eliceiri K.W. Campagnola P.J. Structural changes in mixed Col I/Col V collagen gels probed by SHG microscopy: implications for probing stromal alterations in human breast cancer.Biomed Opt Express. 2011; 2: 2307-2316Crossref PubMed Scopus (63) Google Scholar, 18Provenzano P.P. Eliceiri K.W. Campbell J.M. Inman D.R. White J.G. Keely P.J. Collagen reorganization at the tumor-stromal interface facilitates local invasion.BMC Med. 2006; 4: 38Crossref PubMed Scopus (1185) Google Scholar, 19Bredfeldt J.S. Liu Y. Conklin M.W. Keely P.J. Mackie T.R. Eliceiri K.W. Automated quantification of aligned collagen for human breast carcinoma prognosis.J Pathol Inform. 2014; 5 (eCollection 2014): 28Crossref PubMed Google Scholar, 20Tilbury K. Lien C.H. Chen S.J. Campagnola P.J. Differentiation of Col I and Col III isoforms in stromal models of ovarian cancer by analysis of second harmonic generation polarization and emission directionality.Biophys J. 2014; 106: 354-365Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 21Maller O. Hansen K.C. Lyons T.R. Acerbi I. Weaver V.M. Prekeris R. Tan A.C. Schedin P. Collagen architecture in pregnancy-induced protection from breast cancer.J Cell Sci. 2013; 126: 4108-4110Crossref PubMed Scopus (73) Google Scholar, 22Levental K.R. Yu H. Kass L. Lakins J.N. Egeblad M. Erler J.T. Fong S.F. Csiszar K. Giaccia A. Weninger W. Yamauchi M. Gasser D.L. Weaver V.M. Matrix crosslinking forces tumor progression by enhancing integrin signaling.Cell. 2009; 139: 891-906Abstract Full Text Full Text PDF PubMed Scopus (2700) Google Scholar, 23Lopez J.I. Kang I. You W.K. McDonald D.M. Weaver V.M. In situ force mapping of mammary gland transformation.Integr Biol (Camb). 2011; 3: 910-921Crossref PubMed Scopus (213) Google Scholar, 24Conklin M.W. Keely P.J. Why the stroma matters in breast cancer: insights into breast cancer patient outcomes through the examination of stromal biomarkers.Cell Adh Migr. 2012; 6: 249-260Crossref PubMed Scopus (153) Google Scholar In fact, targeting collagen in the tumor stroma can effectively reduce pulmonary metastasis in breast cancer models.4Lyons T.R. O'Brien J. Borges V.F. Conklin M.W. Keely P.J. Eliceiri K.W. Marusyk A. Tan A.C. Schedin P. Postpartum mammary gland involution drives progression of ductal carcinoma in situ through collagen and COX-2.Nat Med. 2011; 17: 1109-1115Crossref PubMed Scopus (266) Google Scholar, 6Gilkes D.M. Chaturvedi P. Bajpai S. Wong C.C. Wei H. Pitcairn S. Hubbi M.E. Wirtz D. Semenza G.L. Collagen prolyl hydroxylases are essential for breast cancer metastasis.Cancer Res. 2013; 73: 3285-3296Crossref PubMed Scopus (207) Google ScholarIn contrast to the documented procarcinogenic effects of Col1, a gap in knowledge exists regarding the role for the related fibrillar type III collagen (Col3) in breast cancer. Col3 plays a critical function in tissue and organ maintenance, in part through its ability to regulate Col1 fibrillogenesis.25Liu X. Wu H. Byrne M. Krane S. Jaenisch R. Type III collagen is crucial for collagen I fibrillogenesis and for normal cardiovascular development.Proc Natl Acad Sci U S A. 1997; 94: 1852-1856Crossref PubMed Scopus (421) Google Scholar We have identified a role for Col3, distinct from Col1, in promoting a regenerative wound-healing response after cutaneous injury. Specifically, we showed that Col3 promotes reepithelialization and suppresses scar formation through its ability to limit the density of myofibroblasts, the key cellular effectors of scar formation/fibrosis, within wound granulation tissue.26Volk S.W. Wang Y. Mauldin E.A. Liechty K.W. Adams S.L. Diminished type III collagen promotes myofibroblast differentiation and increases scar deposition in cutaneous wound healing.Cells Tissues Organs. 2011; 194: 25-37Crossref PubMed Scopus (158) Google Scholar Given the long-standing view of cancer as wounds that do not heal and the more recently described wound healing, chronic fibrosis, and cancer progression triad,27Dvorak H.F. Tumors: wounds that do not heal: similarities between tumor stroma generation and wound healing.N Engl J Med. 1986; 315: 1650-1659Crossref PubMed Scopus (3482) Google Scholar, 28Rybinski B. Franco-Barraza J. Cukierman E. The wound healing, chronic fibrosis, and cancer progression triad.Physiol Genomics. 2014; 46: 223-244Crossref PubMed Scopus (143) Google Scholar we hypothesized that diminished Col3 in the tumor microenvironment would drive aggressive cancer behaviors and promote metastasis. Because many of the same cells and extracellular proteins (eg, collagens and growth factors) that cause wound healing to go awry also drive aggressive cancer behavior,27Dvorak H.F. Tumors: wounds that do not heal: similarities between tumor stroma generation and wound healing.N Engl J Med. 1986; 315: 1650-1659Crossref PubMed Scopus (3482) Google Scholar, 28Rybinski B. Franco-Barraza J. Cukierman E. The wound healing, chronic fibrosis, and cancer progression triad.Physiol Genomics. 2014; 46: 223-244Crossref PubMed Scopus (143) Google Scholar it is noteworthy that myofibroblasts and a fibrotic healing response characteristic of Col3-deficient wounds have both been implicated in breast cancer metastasis.29Mehner C. Radisky D.C. Triggering the landslide: the tumor-promotional effects of myofibroblasts.Exp Cell Res. 2013; 319: 1657-1662Crossref PubMed Scopus (30) Google Scholar, 30Yamashita M. Ogawa T. Zhang X. Hanamura N. Kashikura Y. Takamura M. Yoneda M. Shiraishi T. Role of stromal myofibroblasts in invasive breast cancer: stromal expression of alpha-smooth muscle actin correlates with worse clinical outcome.Breast Cancer. 2012; 19: 170-176Crossref PubMed Scopus (143) Google Scholar, 31Atula S. Grenman R. Syrjanen S. Fibroblasts can modulate the phenotype of malignant epithelial cells in vitro.Exp Cell Res. 1997; 235: 180-187Crossref PubMed Scopus (66) Google Scholar Consistent with our hypothesis that Col3 loss promotes aggressive breast cancer behaviors, recent data show that a robust stromal response that includes increased Col3 expression correlates with improved survival of breast cancer patients.32Beck A.H. Espinosa I. Gilks C.B. van de Rijn M. West R.B. The fibromatosis signature defines a robust stromal response in breast carcinoma.Lab Invest. 2008; 88: 591-601Crossref PubMed Scopus (87) Google ScholarHerein, we present in vitro and in vivo evidence that Col3 plays an important role in suppressing breast cancer growth and metastasis, using murine (4T1) and human (MDA-MB-231) metastatic triple-negative breast cancer cell lines, a cancer type with limited effective treatment options. Specifically, we show that Col3 is a critical regulator of tumor stroma organization and that diminished levels of Col3 promote tumor cell survival, proliferation, and key processes in metastasis (adhesion, invasion, and migration). Collectively, our findings show that Col3 suppresses procarcinogenic behavior of breast cancer cells to limit primary tumor growth and metastasis and suggest that a Col3-deficient tumor microenvironment promotes metastasis, in part, through the impact of Col3 on stromal organization.Materials and MethodsCol3-Deficient MiceAnimal use and care were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania (Philadelphia) and followed guidelines set forth in the NIH Guide for the Care and Use of Laboratory Animals.33Committee for the Update of the Guide for the Care and Use of Laboratory AnimalsNational Research CouncilGuide for the Care and Use of Laboratory Animals: Eighth Edition. National Academies Press, Washington, DC2011Crossref Google Scholar All mice for this study were generated in a colony established at the University of Pennsylvania from breeder pairs of Col3a1 heterozygous (Col3+/−) mice originally purchased from Jackson Laboratories (Bar Harbor, ME). These mice had been generated by homologous recombination by replacement of the promoter region and first exon of the Col3 gene with a 1.8-kb PGKneo cassette,25Liu X. Wu H. Byrne M. Krane S. Jaenisch R. Type III collagen is crucial for collagen I fibrillogenesis and for normal cardiovascular development.Proc Natl Acad Sci U S A. 1997; 94: 1852-1856Crossref PubMed Scopus (421) Google Scholar generating a global knockout. Animals were genotyped for Col3 by PCR analysis of DNA extracted from tail biopsy specimens and were microchipped for identification (Allflex FDX-B transponders; Allflex USA, Inc., Dallas, TX).26Volk S.W. Wang Y. Mauldin E.A. Liechty K.W. Adams S.L. Diminished type III collagen promotes myofibroblast differentiation and increases scar deposition in cutaneous wound healing.Cells Tissues Organs. 2011; 194: 25-37Crossref PubMed Scopus (158) Google Scholar, 34Volk S.W. Shah S.R. Cohen A.J. Wang Y. Brisson B.K. Vogel L.K. Hankenson K.D. Adams S.L. Type III collagen regulates osteoblastogenesis and the quantity of trabecular bone.Calcif Tissue Int. 2014; 94: 621-631Crossref PubMed Scopus (42) Google ScholarAll mice in the Col3 colony, regardless of the study they were used for, were surveyed routinely for general health and pathology, including gross evidence of tumor development. Once a trend was recognized for an increase in tumor formation in Col3+/− mice, all mice dying spontaneously or euthanized at >1 year of age had a gross necropsy performed, and a biopsy of any abnormal masses was performed. Histopathological diagnoses of tumor biopsy specimens were made by a board-certified veterinary pathologist (E.A.M.).Cell Culture and Tumor Generation4T1 and 4T1–green fluorescent protein (GFP) cell lines (obtained from Dr. Lalage Wakefield at the National Cancer Institute, Bethesda, MD, in 201035Aslakson C.J. Miller F.R. Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor.Cancer Res. 1992; 52: 1399-1405PubMed Google Scholar) and MDA-MB-231 cells [obtained from Dr. Meenhard Herlyn (Wistar Institute, Philadelphia, PA) in 201236Peng H. Talebzadeh-Farrooji M. Osborne M.J. Prokop J.W. McDonald P.C. Karar J. Hou Z. He M. Kebebew E. Orntoft T. Herlyn M. Caton A.J. Fredericks W. Malkowicz B. Paterno C.S. Carolin A.S. Speicher D.W. Skordalakes E. Huang Q. Dedhar S. Borden K.L. Rauscher 3rd, F.J. LIMD2 is a small LIM-only protein overexpressed in metastatic lesions that regulates cell motility and tumor progression by directly binding to and activating the integrin-linked kinase.Cancer Res. 2014; 74: 1390-1403Crossref PubMed Scopus (22) Google Scholar] were authenticated by morphology, growth characteristics, and biological behavior, tested for Mycoplasma, and frozen. Cells were cultured for <4 months. All cells were cultured in growth media: Dulbecco's modified Eagle’s medium (Glutamax; Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA) and antibiotics (100 U/mL penicillin and 100 g/mL streptomycin). GFP expression in the 4T1-GFP cell line was preserved with inclusion of 50 μg/mL G418 (Geneticin, Life Technologies, Carlsbad, CA) in the growth media.For orthotopic tumor implantation, 0.1 or 0.5 × 106 4T1 cells [in 0.1 mL sterile phosphate-buffered saline (PBS)] were injected s.c. into the right fourth mammary fat pad of anesthetized mice (aged 8 to 20 weeks). Tumor volume was calculated using the following formula: V = (L × W2)/2.37Egunsola A.T. Zawislak C.L. Akuffo A.A. Chalmers S.A. Ewer J.C. Vail C.M. Lombardo J.C. Perez D.N. Kurt R.A. Growth, metastasis, and expression of CCL2 and CCL5 by murine mammary carcinomas are dependent upon Myd88.Cell Immunol. 2012; 272: 220-229Crossref PubMed Scopus (19) Google ScholarTissue ProcessingPrimary tumors, mammary fat pads, and lungs were collected and fixed in Prefer Fixative (Anatech LTD, Battle Creek, MI). Before fixation, lungs were perfused through the heart and trachea. Tissues were paraffin embedded and processed, and serial sections (4 μm thick) were stained with hematoxylin and eosin (H&E), as previously described.26Volk S.W. Wang Y. Mauldin E.A. Liechty K.W. Adams S.L. Diminished type III collagen promotes myofibroblast differentiation and increases scar deposition in cutaneous wound healing.Cells Tissues Organs. 2011; 194: 25-37Crossref PubMed Scopus (158) Google ScholarAnalysis of Pulmonary MetastasesAfter perfusion and fixation, gross lung metastases in mice with orthotopic 4T1 tumors were counted. All lung lobes were bisected lengthwise through the main stem bronchi.38Leustik M. Doran S. Bracher A. Williams S. Squadrito G.L. Schoeb T.R. Postlethwait E. Matalon S. Mitigation of chlorine-induced lung injury by low-molecular-weight antioxidants.Am J Physiol Lung Cell Mol Physiol. 2008; 295: L733-L743Crossref PubMed Scopus (82) Google Scholar Quantitation of metastasis on H&E-stained slides containing a cross section of all five lung lobes was performed by a pathologist (E.A.M.). Lung tumor burden was quantitated using ImageJ software version 1.48 (NIH, Bethesda, MD), as previously described.39Santos A.M. Jung J. Aziz N. Kissil J.L. Pure E. Targeting fibroblast activation protein inhibits tumor stromagenesis and growth in mice.J Clin Invest. 2009; 119: 3613-3625Crossref PubMed Scopus (293) Google Scholar To examine how Col3 in the metastatic niche regulates metastasis, tail vein injections and analysis of pulmonary colonization were performed. Specifically, 7- to 11-week-old male mice were injected with 1.0 × 106 4T1-GFP cells (via tail vein). After 24 hours, the mice were sacrificed, and the lungs were perfused with PBS. A single-cell suspension was collected after treating the lungs with a mixture of collagenases. Cells were stained with propidium iodine and analyzed via flow cytometry using a LSRFortessa (BD Biosciences, Bedford, MA). The percentage of live (propidium iodine–negative) 4T1 (GFP-positive) cells within the lung was calculated.Tissue Immunohistochemistry and ImmunofluorescenceSections from fixed, paraffin-embedded tissues were mounted onto charged glass slides. After deparaffinization and rehydration, antigen retrieval was performed by citrate buffer boiling or incubation with proteinase K (20 μg/mL in Tris-ethylenediaminetetraacetic acid buffer for Col3 staining).For immunohistochemistry, sections were blocked in 3% H2O2, PBS containing 1% bovine serum albumin (A5611; Sigma-Aldrich, St. Louis, MO), 10% goat serum, avidin blocking solution, and biotin blocking solution (Avidin Blocking Kit; Vector Laboratories Inc., Burlingame, CA). For immunofluorescence, sections were blocked in PBS containing 5% bovine serum albumin, 5% goat serum, and 0.05% Tween-20 (Bio-Rad, Hercules, CA). Slides were incubated with antibodies directed against Col3 (ab7778; Abcam, Cambridge, MA), Ki-67 (ab15580; Abcam), active caspase 3 (9664; Cell Signaling, Danvers, MA), or α-smooth muscle actin (α-SMA; ab5694; Abcam). Slides were then incubated in secondary antibody, biotin-goat anti-rabbit IgG (BA 1000; Vector Laboratories Inc.), then incubated in tertiary antibody (ABC elite; Vector Laboratories Inc.) and incubated in DAB+ Substrate (Dako, Carpinteria, CA) until brown color developed. Slides were counterstained in hematoxylin, before dehydration and mounting.For immunofluorescence, sections were incubated with an Alexa Fluor 488 goat anti-rabbit antibody (Invitrogen, Grand Island, NY) and mounted in medium containing DAPI (Vector Laboratories Inc.). All slides were viewed with an Olympus BX51 microscope (Olympus, Melville, NY), and digital images were obtained using a constant exposure threshold. For active caspase 3 and α-SMA staining quantification, ImageJ software was used to measure the percentage area of the image that contained positive staining. The proliferative index was calculated using ImageJ software (percentage of Ki-67–positive nuclei/total nuclei).Quantitative Real-Time PCRmRNA expression analysis was performed as previously described.34Volk S.W. Shah S.R. Cohen A.J. Wang Y. Brisson B.K. Vogel L.K. Hankenson K.D. Adams S.L. Type III collagen regulates osteoblastogenesis and the quantity of trabecular bone.Calcif Tissue Int. 2014; 94: 621-631Crossref PubMed Scopus (42) Google Scholar Briefly, RNA was extracted from fibroblasts, 4T1 cells, and mouse tissues [mammary fat pads and lungs from tumor-naïve mice and tumors (14 days after orthotopic injection of 4T1 cells)], cDNA was generated, and Col1 and Col3 expression was compared to glyceraldehyde-3-phosphate dehydrogenase as the endogenous control. For mouse tissues, lungs from young (16 to 18 weeks old) and aged (92 to 94 weeks old), and fat pads and tumors from young (10 to 24 weeks old), mice were used.Fibroblast Isolation and CultureEmbryonic fibroblasts were harvested and genotyped as described previously.26Volk S.W. Wang Y. Mauldin E.A. Liechty K.W. Adams S.L. Diminished type III collagen promotes myofibroblast differentiation and increases scar deposition in cutaneous wound healing.Cells Tissues Organs. 2011; 194: 25-37Crossref PubMed Scopus (158) Google Scholar Fibroblasts were cultured and passaged (passage 6 or less), as described for 4T1 cells, including the addition of l-ascorbic acid (A8960; Sigma-Aldrich) to the growth media to ensure secretion of a collagen-rich matrix.Generation of Fibroblast-Derived MatricesDecellularized matrices were generated, as previously described,40Beacham D.A. Amatangelo M.D. Cukierman E. Preparation of extracellular matrices produced by cultured and primary fibroblasts.Curr Protoc Cell Biol. 2007; (Chapter 10:Unit 10.9)PubMed Google Scholar using E18.5 embryonic fibroblasts. After 5 to 8 days in culture, the matrices were decellularized and were either stored in PBS at 4°C or used immediately for experiments.Proliferation AssaysFibroblast-derived matrices were generated in 96-well plates (1.0 × 104 fibroblasts per well), as described above. 4T1 (1.5 × 104) or MDA-MB-231 (2.5 × 104) cells were plated and cultured on these decellularized matrices in growth media for 6 hours before changing to serum-free media overnight. Proliferation was measured using a bromodeoxyuridine, 96-well, enzyme-linked immunosorbent assay–based assay (QIA58; EMD Millipore, Billerica, MA), using a Varioskan Flash plate reader (Thermo Fisher Scientific, Waltham, MA).Analysis of Apoptotic Cells4T1 or MDA-MB-231 cells (1.0 × 104) were plated onto fibroblast-derived matrix-coated coverslips in 24-well plates, cultured in growth media for 16 hours, and then switched to serum-free media for 48 hours. After fixation with 4% paraformaldehyde, coverslips were incubated with an antibody directed against active caspase 3 (ab2302; Abcam) and subsequently with an Alexa Fluor 488 donkey anti-rabbit antibody (Invitrogen), before mounting in medium containing DAPI. Fluorescence was viewed as described above for sections.Adhesion AssaysFor assessment of Col3 modulation of 4T1 morphology and adhesion, fibroblasts (5.0 × 104) were plated and cultured in 24-well plates for 48 hours in growth media before subsequent seeding of 4T1-GFP cells (8.0 × 105). 4T1-GFP cells were allowed to adhere for 2 hours in growth media or remain in culture for 48 hours in serum-free media to assess morphology, then fixed and imaged. For adhesion, GFP intensity was quantitated using ImageJ software. For adhesion in collagen-coated wells, 24-well plates were coated with 0.5 μg/cm2 human placenta-derived (hP) collagens hPCol1 (354243; BD Biosciences), hPCol3 (354244; BD Biosciences), or a 50:50 (0.5 μg/cm2 total collagen) or 100:50 (0.75 μg/cm2 total collagen) mixture of both. 4T1 cells (5.0 × 105) were plated in growth media and allowed to adhere for 2 hours. Attached cells were stained with crystal violet, and OD 570 was measured using a Varioskan plate reader. For adhesion to a substratum with stiffness similar to mammary fat pad,41Otranto M. Sarrazy V. Bonte F. Hinz B. Gabbiani G. Desmouliere A. The role of the myofibroblast in tumor stroma remodeling.Cell Adh Migr. 2012; 6: 203-219Crossref PubMed Scopus (155) Google Scholar hydrogels (6 kPa) were generated as described previously42Olsen A.L. Bloomer S.A. Chan E.P. Gaca M.D. Georges P.C. Sackey B. Uemura M. Janmey P.A. Wells R.G. Hepatic stellate cells require a stiff environment for myofibroblastic differentiation.Am J Physiol Gastrointest Liver Physiol. 2011; 301: G110-G118Crossref PubMed Scopus (232) Google Scholar on 12-mm coverslips in 24-well plates. The hydrogels were coated with hPCol1 and hPCol3 at 0.03 mg/mL or a 50:50 mixture of both. 4T1-GFP cells were" @default.
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• W2011953153 title "Type III Collagen Directs Stromal Organization and Limits Metastasis in a Murine Model of Breast Cancer" @default.
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