Next, we used these double transgenic mice as donors and recipients in adoptive BM transplantations as described above for PyMT-GFP mice (Fig. Unlike resident CAFs, BM-derived CAFs do not express PDGFRα, and their recruitment resulted in a decrease in the percentage of PDGFRα-expressing CAFs. (G) FACS analysis of PDGFRα in MSCs incubated with Met-1 CM. As immature cells, fibroblasts can differentiate into other connective … Other members of the connective tissue family include bone, cartilage, fat, and smooth muscle cells. Strikingly, we found significant differences between resident and BM-derived CAFs: BM-derived CAFs were more efficient in inducing angiogenesis, which was evident even by macroscopic examination (Fig. For specific breast cancer subtypes, patients were stratified by their expression of PR, ER, HER2, or lack of expression of all three and analyzed for survival as above with GraphPad. There are three main forms: essential thrombocythaemia, polycythaemia vera and myelofibrosis in which the overproduced cell types are platelets, red blood cells and fibroblasts, respectively. Error bars represent SD of technical repeats. Cancer-associated fibroblasts (CAFs) are highly prominent in breast tumors, but their functional heterogeneity and origin are still largely unresolved. We demonstrated that the molecular mechanism by which BM-derived CAFs promote angiogenesis is mediated by up-regulation of the secreted glycoprotein Clusterin, shown to affect tumor cell growth, angiogenesis, and resistance to therapy (Fu et al., 2013; Koltai, 2014; Tellez et al., 2016; Wang et al., 2018). Stromal cells secrete extracellular matrix components such as collagens and can remodel the ECM. Thus, BM-derived CAFs have distinct gene expression and tumor-promoting functions, and their enhancement of tumor growth is mediated, at least in part, by up-regulation of the pro-angiogenic factor Clusterin. For I and J, multiple fields from at least three mice were analyzed. Interestingly, a previous study of inflammation-induced gastric cancer suggested that tumor-derived paracrine signaling contributes to modification of the MSC niche within the BM (Quante et al., 2011). The authors suggested that human dermal fibroblasts represent a convenient cell source for bone and cartilage tissue engineering. Bell performed bioinformatics analysis, L. Leider-Trejo, D. Grisaru, and M. Milyavsky were involved in data analysis and discussions; C. Levy and H.L. Both mouse lines (Col1α-DsRed and Col1α-YFP) were backcrossed for 10 generations to the FVB/n background to be syngeneic with the MMTV-PyMT model. with 2.5 × 106 unfractionated BM cells harvested aseptically from flushed femur and tibia of age-matched FVB/n-GFP, MMTV-PyMT;GFP, Col1α-DsRed/YFP, or PyMT;Col1α-DsRed/YFP female mice. 2 wk following transplantation, Met-1 cells were injected into the right inguinal mammary gland of the transplanted mice. Bars, 100 µm. To study the possible influence of the BMT procedure itself on tumor growth, transplanted mice were followed for weight and tumor development in comparison to age-matched control FVB/n and MMTV-PyMT mice. Tissue sections were incubated overnight at 4°C with the following anti-mouse antibodies: FSP1 (Abcam; ab27957), αSMA (Sigma-Aldrich; F3777), Vimentin (Millipore; AB1620), PDGFRα (Santa Cruz; sc-101569), GFP (Rockland; 600-101-215), RFP (Rockland; 600-401-379), F4/80 (AbD Serotec; MCA497), Meca32 (BD; 550563) and cleaved caspase-3 (Abcam; Ab32042). Experiment was repeated twice, n ≥ 3 mice per group for each experiment. n = 4 mice at each stage; 4 high-power fields were analyzed for each mouse (24 high-power fields total). Alternatively, for in vitro experiments, total RNA was isolated from cell pellets using PureLinkTM RNA Mini kit (Invitrogen; 12183018A). (H) Migration transwell assay of Met-1 mammary tumor cells incubated with tumor-activated NMFs (a-NMFs) or MSCs (a-MSCs) for 24 h. Representative images of 24 fields analyzed from duplicate wells. Human umbilical cord blood (CB) has attracted much attention as a reservoir for functional hematopoietic stem and progenitor cells, and, recently, as a source of blood-borne fibroblasts (CB-BFs). Fibroblast, the principal active cell of connective tissue. Together, these biosynthetic, pro-inflammatory, contractile, and adhesive functions enable fibroblasts to execute effective wound healing. Representative of two independent experiments. (B) Venn diagrams of the 40 most highly expressed genes in each cell population. Tumors were measured every 2–3 d with a caliper, and tumor volumes were calculated using the formula X2 × Y × 0.52 (X = smaller diameter; Y = larger diameter). JingyinYan. PDGFRα was previously shown to be a robust marker of fibroblasts (Erez et al., 2010; Driskell et al., 2013; Sharon et al., 2013; Ruffell et al., 2014). The number of fibroblasts increases in the healing wound while the number of inflammatory cells decreases. Control mice that were irradiated but not transplanted with BM died within 2 wk of irradiation. (B–G) PDGFRα is a marker of resident fibroblasts. Bars, 50 µm. Bone tissue degeneration is an urgent clinical issue, making it a subject of intensive research. Furthermore, analysis of blood vessel density (Meca32 staining) indicated that tumors that contained both resident and BM-derived CAFs were significantly more vascularized than tumors with resident CAFs only (Fig. Tumor growth was measured for 24 d after injection. Thus, the decrease in the percentage of PDGFRα+ CAFs in breast tumors and lung metastases is, at least partially, a result of recruitment of a PDGFRα− fibroblastic cell population from the BM. 1 G). Surprisingly, the inverse analysis revealed an unexpected decrease in the percentage of PDGFRα+ fibroblasts within the growing αSMA+ population (Fig. Yael Raz, Noam Cohen, Ophir Shani, Rachel E. Bell, Sergey V. Novitskiy, Lilach Abramovitz, Carmit Levy, Michael Milyavsky, Leonor Leider-Trejo, Harold L. Moses, Dan Grisaru, Neta Erez; Bone marrow–derived fibroblasts are a functionally distinct stromal cell population in breast cancer. This research was supported by grants to N. Erez from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement no. At 70% confluency, cells were transfected with Accell Delivery Media (GE Dharmacon; B-005000) supplemented with 1 µM Accell SMARTpool mouse Clusterin siRNA (GE Dharmacon; E-043966-00) or Accell Control Pool nontargeting siRNA (GE Dharmacon; D-001910-10) for 48 h. Accell SMARTpool contains a mixture of four siRNAs targeting one gene and provides extended duration of gene knockdown with only minimal effects on cell viability and the innate immune response. ****, P < 0.0001, two-tailed Mann-Whitney test. OGX-11 was also tested in clinical trials in metastatic prostate cancer (Beer et al., 2017), and other trials are ongoing. Strikingly, macroscopic analysis of the plugs indicated that plugs injected with BM-derived CAFs in which Clusterin was knocked down were less vascularized than control plugs (Fig. 1 wk after injection the plugs were dissected out and embedded in OCT for histological analysis. Ca, carcinoma; MG, mammary gland. Human fibroblasts, also known as stromal cells, have a wide range of fascinating cellular functions and practical applications. Collectively, these observations suggest that in addition to the population of resident PDGFRα+ CAFs, there is a distinct subpopulation of PDGFRα− CAFs. Following biomaterial implantation, fibroblasts undergo a response known as ‘activation,’ characterized by a transition of quiescent cells into myofibroblast-like phenotype. YFP+ cells are shown in green. S5 E for details). Following 6-h starvation, cells were incubated with conditioned media from PDGFRα+ fibroblasts, PDGFRα− incubated with nontargeting siRNA or PDGFRα− incubated with Clusterin siRNA. They orchestrate the continual production and turnover of the pulmonary extracellular matrix, the correct organization of which is vital for efficient gas exchange. A major obstacle in characterizing CAFs is the lack of a single specific surface marker that can be used to detect and isolate all fibroblasts. Analysis of the expression data in human breast cancer revealed a significant decrease in the expression of PDGFRα (Fig. *, P = 0.05; one-tailed Mann-Whitney test. Bars, 30 µm. CAFs are vastly heterogeneous and are comprised of several subpopulations with diverse origins, including activated myofibroblasts (characterized by α-smooth muscle actin [αSMA] expression), reprogrammed local tissue fibroblasts (Sharon et al., 2015) and adipocyte-derived CAFs (Bochet et al., 2013). However, in these studies the expression of Clusterin was analyzed in total tumor tissue sections, without specifically analyzing the expression in CAFs. Representative images of multiple fields analyzed from four mice in two independent experiments. 1, A and B). A small population of CD45+Col1α+ cells was found in BM, likely representing fibrocytes, which are of hematopoietic origin (Bucala et al., 1994; Fig. In these settings, uncontrolled fibroblast activation and proliferation or, conversely, the loss of fibroblast number or function, contribute to the initiation and progression of abnormal lung repair. Moreover, there was almost no expression of fibroblastic markers in YFP− cells. Fibroblasts are important cells that secrete a diverse range of paracrine factors that affect cell and tissue homeostasis. These roles highlight the versatility and indispensability of fibroblasts to the maintenance of normal lung physiology. Sorting was done with BD FACSAria II. S1 shows gating strategy of FACS experiments presented in Fig. To further analyze the subpopulation of BM-derived fibroblasts, we analyzed cell populations isolated from mammary tumors of BM transplanted mice by FACS. Similarly, a distinct population of GFP+PDGFRα– (BM-derived) fibroblasts was recruited to lung metastases. When cells were at 80% confluency, plates were washed twice with PBS and fresh medium was applied. 2 M). A fibroblast is a type of biological cell that synthesizes the extracellular matrix and collagen , produces the structural framework (stroma) for animal tissues, and plays a critical role in wound healing. Fibroblasts are versatile spindle-shaped cells distributed in different parenchymal tissues that are capable of both ECM synthesis and resorption. (D–I) PDGFRα+-resident CAFs and PDGFRα− BM-derived CAFs have distinct tumor-promoting functions. We next set out to investigate the distinct functional role of BM-derived CAFs in mammary tumor growth in vivo. **, P = 0.022; two-tailed Mann-Whitney test. Following BM transplantation, resident and BM-derived CAFs from mammary tumors and lungs bearing macrometastases of recipient mice were tested for the expression of 561 immunology-related genes using the NanoString nCounter gene expression panel. Data analysis was done with BD FACSDiva software (BD Biosciences), CytExpert 2.0 (Beckman Coulter, Inc.) or the Kaluza Flow Analysis software (Beckman Coulter, Inc.). Error bars represent SEM. Intracardiac fibroblasts, but not bone marrow derived cells, are the origin of myofibroblasts in myocardial infarct repair Cardiovasc Pathol . The percentage of PDGFRα+ fibroblasts was quantified from the total number of fibroblasts for each section. 4, F and G; and Fig. 6, F and G). Although MEFs are the most common feeder cell type used with hPSCs, human fibroblasts can also be used. Although unstimulated fibroblasts are biosynthetically quiescent, under the influence of appropriate extra-cellular cues they secrete ECM macromolecules and proteolytic enzymes, growth factors, cytokines and chemokines; adhere to and contract connective … Analysis was performed with BD FACSAria II. Patients were divided into two groups of high and low expression levels using the median expression. Single cell suspensions of mammary glands were prepared as we previously described (Sharon et al., 2013). (J) Immunofluorescent staining as in I in normal lungs from FVB/n Col1α-YFP recipients or in lung macrometastases from PyMT;Col1α-YFP recipients. Thus, tumor-specific mechanisms of recruitment could be operative in different tumor types. In all analyses expression results were normalized to GAPDH and mGUS. RQ (2-ΔCt) was calculated. Expression of selected genes was validated by qRT-PCR. BM transplantations were repeated five times (n = 2–4 mice in each cohort). Upon recruitment, they acquire specific functional CAF characteristics including the ability to attract immune cells and induce angiogenesis. Terminology: The appearance of "blast" in a cell name normally indicates an embryonic cell that transforms into a mature cell type (e.g., neuroblast, myoblast). Analysis of BM smears from transplanted mice confirmed the repopulation of recipient BM with donor cells (Fig. 2, and qRT-PCR analysis of resident and BM-derived fibroblasts. The C166 endothelial cell line was purchased from the ATCC. Pre-processed and normalized RNA-seq gene expression in 10 diverse cancer types were obtained (see Fig. Analysis or sorting was done with BD FACSAria II, BD FACSAria Fusion or CytoFLEX Flow Cytometer (Beckman Coulter, Inc.). ectopic calcification, fibroblasts are concerned as principal can-didates.33 Dermal fibroblasts treated with pro-calcifying medium for 3 weeks exhibited evident calcium deposits.34,35 Interestingly, Federica et al found that dermal fibroblasts derived from PXE patients … (F) qRT-PCR of PDGFRα+ lung CAFs, FACS sorted from a pool of end stage MMTV-PyMT mice (n = 5) and normal lung fibroblasts pooled from normal mice (n = 6). The relative contribution of BM-derived fibroblasts to the overall population of CAFs may be tumor type-specific (Arina et al., 2016). Fibroblasts at wound sites have prominent cytoplasmic actin and myosin fibers and are sometimes referred to as myofibroblasts. FACS sorting of YFP+ cells from mammary tumors followed by qRT-PCR confirmed that YFP+ cells express multiple fibroblast markers but do not express epithelial, endothelial, or immune cell markers. Fibrocytes are BM-derived cells of hematopoietic origin with fibroblastic characteristics, which express hematopoietic markers (CD45 and CD34; Bucala et al., 1994). n = 2 mice in the control group and 6 mice in the siClusterin group. 1 J). Using bone marrow transplantation of transgenic mice that express GFP driven by collagen 1A1 promoter, we have shown that bone marrow-derived hematopoietic fibroblasts migrate into the kidney, proliferate, and differentiate into α-SMA + myofibroblasts (Xia et al., 2014b). 1 H and Fig. The authors would like to thank Dr. Irena Shur and the Sackler School of Medicine Interdepartmental Core Facility (SICF) for help with imaging, FACS, and PCR analyses and Elad Yana for graphical assistance. (l) Quantification of K. Multiple fields (at least six) from four different experiments were analyzed. Error bars represent SD or SEM as indicated. ScienceDirect ® is a registered trademark of Elsevier B.V. ScienceDirect ® is a registered trademark of Elsevier B.V. URL: https://www.sciencedirect.com/science/article/pii/B9780123740014000158, URL: https://www.sciencedirect.com/science/article/pii/B0123708796001563, URL: https://www.sciencedirect.com/science/article/pii/B0122267656002486, URL: https://www.sciencedirect.com/science/article/pii/B9780123854735000023, URL: https://www.sciencedirect.com/science/article/pii/B9780128035818098283, URL: https://www.sciencedirect.com/science/article/pii/B9780080552941001008, URL: https://www.sciencedirect.com/science/article/pii/B9781416002468500097, URL: https://www.sciencedirect.com/science/article/pii/B9780128031025000112, URL: https://www.sciencedirect.com/science/article/pii/B9780323316965000838, Polyphenols: Prevention and Treatment of Human Disease (Second Edition), 2018, Lynne A. Murray, ... Geoffrey J. Laurent, in, Encyclopedia of Immunology (Second Edition), Preparation of Mouse Embryonic Fibroblast Feeder Cells, Trevor R. Leonardo, ... Suzanne E. Peterson, in, THE IMMUNE SYSTEM AND THE INFLAMMATORY RESPONSE, Textbook of Pediatric Rheumatology (Fifth Edition), Mesenchymal Stem Cell Transition to Tumor-Associated Stromal Cells Contributes to Cancer Progression, Mesenchymal Stromal Cells as Tumor Stromal Modulators, Kelley and Firestein's Textbook of Rheumatology (Tenth Edition), Journal of Molecular and Cellular Cardiology. To that end, we isolated repopulating hematopoietic stem cells (HSCs; c-Kit+Lin−Sca-1+CD34−CD45+) and MSCs (c-Kit+Lin−Sca-1+CD34−CD45−) from BM of Col1α-DsRed female mice and used them for adoptive BM transplantations into irradiated Col1α-YFP recipient mice. Their cytoplasm is basophilic, so there will be quite a lot of purplish blue staining of the cytoplasm, using the H&E stain. DsRed expression was significantly higher in tumors from mice transplanted with MSCs, as compared with mice transplanted with HSCs only, indicating the recruitment of BM-derived fibroblasts of mesenchymal origin in these mice (Fig. (F) Immunofluorescence of cleaved caspase-3 in tumor sections from transplanted mice. Moreover, resident CAFs and BM-derived CAFs were distinct in their ability to induce angiogenesis and to recruit macrophages. Human fibroblasts provide a number of advantages over MEFs in that they can be propagated for many more passages than MEFs before senescing. In addition to matrix formation, immune functions have also been ascribed to fibroblasts, including antigen presentation and the secretion of bioactive molecules. Thus, our findings that PDGFRα expression distinguishes two functionally unique CAF populations may have implications for patient stratification and tailored therapeutics in breast cancer. They are the primary source of extracellular matrix (ECM) proteins, which, in addition to providing a scaffold for cells, play key roles in determining cell phenotype and function. (A) Scheme of BM transplantation model. Ifor R. Williams, in Encyclopedia of Immunology (Second Edition), 1998. Finally, we review the potential of opportunities arising for better therapeutic intervention strategies targeting fibroblasts that will either halt or potentially reverse fibrosis. Cells were stained using the following antibodies: anti-PDGFRα-PE (eBioscience; 12–1401-81), anti-EpCAM-APC (Miltany Biotec; 130–102-234), anti-CD45-PerCP-Cy5.5 (eBioscience; 45–0451-82), anti-CD45-PE-Cy7 (eBioscience; 25–0451-82), anti-Annexin V-Alexafluor 488 (Invitrogen; A13201), anti-CD34–FITC (eBioscience; 11–0341-82), and anti-αSMA-FITC (Sigma; F3777). (H and J) Immunofluorescence of F4/80 (H) or Meca32 (J) in tumor sections from transplanted mice. A better understanding of the phenotypes of disease-associated fibroblasts may highlight pathways specific to disease pathologies, giving rise to targeted therapeutics. Data from human patients suggest that this unique BM-derived CAF population may have a deleterious effect on survival. P value of ≤0.05 was considered statistically significant for all datasets. (F) Quantification of D and E. Error bars represent SEM. Human fibroblast lines can be obtained from many sources (see above). Cells were grown in DMEM media as above. Adults tend to have less active fibroblasts, called fibrocytes, which have paler stained cytoplasm. Encouraged by these findings we set out to elucidate the role of Clusterin in the pro-angiogenic function of BM-derived CAFs. n = 4. Moreover, only YFP+ resident CAFs, but not DsRed+ BM-derived CAFs, expressed PDGFRα, further validating it is a marker of resident fibroblasts (Fig. S3 C). ****, P < 0.0001 (G); **, P = 0.0041 (I); two-tailed Mann-Whitney test. 6-wk-old Col1α-YFP female mice were lethally irradiated using an x-ray machine (160HF; Philips) at a total dose of 9 Gy. Resident CAFs (EpCAM−CD45−Col1α+PDGFRα+) and BM-derived CAFs (EpCAM−CD45−Col1α+PDGFRα−) were isolated from mammary tumors of PyMT;Col1α-YFP female mice, cultured and treated with siClusterin or siControl for 48 h, after which they were injected in a Matrigel plug with additional siRNA to 6–8-wk-old FVB/n female mice. n = 3 mice; *, P = 0.05; one-tailed Mann-Whitney test. n = 2. We found that BM-derived CAFs do not express PDGFRα, suggesting that other pathways are operative in the signaling between tumor cells and this CAF population. 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