Peroxisome proliferator‐activated receptor gamma (PPARγ) is central to the initiation and propagation of human angiomyolipoma, suggesting its potential as a therapeutic target
2017; Springer Nature; Volume: 9; Issue: 4 Linguagem: Inglês
10.15252/emmm.201506111
ISSN1757-4684
AutoresOren Pleniceanu, Racheli Shukrun, Dorit Omer, Einav Vax, Itamar Kanter, Klaudyna Dziedzic, Naomi Pode‐Shakked, Michal Mark‐Daniei, Sara Pri‐Chen, Yehudit Gnatek, Hadas Alfandary, Nira Varda‐Bloom, Dekel D. Bar‐Lev, Naomi Bollag, Rachel Shtainfeld, Leah Armon, Achia Urbach, Tomer Kalisky, Arnon Nagler, Orit Harari‐Steinberg, Jack L. Arbiser, Benjamin Dekel,
Tópico(s)Renal and related cancers
ResumoResearch Article8 March 2017Open Access Transparent process Peroxisome proliferator-activated receptor gamma (PPARγ) is central to the initiation and propagation of human angiomyolipoma, suggesting its potential as a therapeutic target Oren Pleniceanu Oren Pleniceanu Pediatric Stem Cell Research Institute, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Division of Pediatric Nephrology, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Division of Hematology and Cord Blood Bank, Sheba Medical Center, Ramat Gan, Israel Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Racheli Shukrun Racheli Shukrun Pediatric Stem Cell Research Institute, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Division of Pediatric Nephrology, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Dorit Omer Dorit Omer Pediatric Stem Cell Research Institute, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Division of Pediatric Nephrology, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Search for more papers by this author Einav Vax Einav Vax Pediatric Stem Cell Research Institute, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Division of Pediatric Nephrology, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Itamar Kanter Itamar Kanter Faculty of Engineering, Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, Israel Search for more papers by this author Klaudyna Dziedzic Klaudyna Dziedzic Pediatric Stem Cell Research Institute, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Division of Pediatric Nephrology, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Naomi Pode-Shakked Naomi Pode-Shakked Pediatric Stem Cell Research Institute, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Division of Pediatric Nephrology, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Correction added on 3 April 2017 after first online publication: affiliation 4 has been added for NP-S; affiliations have been corrected from 1,2,3 to 3,4 for AN Search for more papers by this author Michal Mark-Daniei Michal Mark-Daniei Pediatric Stem Cell Research Institute, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Division of Pediatric Nephrology, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Search for more papers by this author Sara Pri-Chen Sara Pri-Chen Pediatric Stem Cell Research Institute, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Division of Pediatric Nephrology, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Search for more papers by this author Yehudit Gnatek Yehudit Gnatek Pediatric Stem Cell Research Institute, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Division of Pediatric Nephrology, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Search for more papers by this author Hadas Alfandary Hadas Alfandary Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Institute of Nephrology, Schneider Children's Medical Center of Israel, Petah Tikva, Israel Search for more papers by this author Nira Varda-Bloom Nira Varda-Bloom Division of Hematology and Cord Blood Bank, Sheba Medical Center, Ramat Gan, Israel Search for more papers by this author Dekel D Bar-Lev Dekel D Bar-Lev Pediatric Stem Cell Research Institute, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Division of Pediatric Nephrology, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Search for more papers by this author Naomi Bollag Naomi Bollag The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel Search for more papers by this author Rachel Shtainfeld Rachel Shtainfeld The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel Search for more papers by this author Leah Armon Leah Armon The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel Search for more papers by this author Achia Urbach Achia Urbach The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel Search for more papers by this author Tomer Kalisky Tomer Kalisky Faculty of Engineering, Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, Israel Search for more papers by this author Arnon Nagler Arnon Nagler Division of Hematology and Cord Blood Bank, Sheba Medical Center, Ramat Gan, Israel Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Correction added on 3 April 2017 after first online publication: affiliation 4 has been added for NP-S; affiliations have been corrected from 1,2,3 to 3,4 for AN Search for more papers by this author Orit Harari-Steinberg Orit Harari-Steinberg Pediatric Stem Cell Research Institute, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Division of Pediatric Nephrology, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Search for more papers by this author Jack L Arbiser Jack L Arbiser Department of Dermatology, Emory University School of Medicine, Atlanta, GA, USA Winship Cancer Institute, Atlanta Veterans Administration Hospital, Atlanta, GA, USA Search for more papers by this author Benjamin Dekel Corresponding Author Benjamin Dekel [email protected] [email protected] orcid.org/0000-0002-3784-8238 Pediatric Stem Cell Research Institute, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Division of Pediatric Nephrology, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Oren Pleniceanu Oren Pleniceanu Pediatric Stem Cell Research Institute, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Division of Pediatric Nephrology, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Division of Hematology and Cord Blood Bank, Sheba Medical Center, Ramat Gan, Israel Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Racheli Shukrun Racheli Shukrun Pediatric Stem Cell Research Institute, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Division of Pediatric Nephrology, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Dorit Omer Dorit Omer Pediatric Stem Cell Research Institute, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Division of Pediatric Nephrology, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Search for more papers by this author Einav Vax Einav Vax Pediatric Stem Cell Research Institute, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Division of Pediatric Nephrology, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Itamar Kanter Itamar Kanter Faculty of Engineering, Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, Israel Search for more papers by this author Klaudyna Dziedzic Klaudyna Dziedzic Pediatric Stem Cell Research Institute, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Division of Pediatric Nephrology, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Naomi Pode-Shakked Naomi Pode-Shakked Pediatric Stem Cell Research Institute, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Division of Pediatric Nephrology, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Correction added on 3 April 2017 after first online publication: affiliation 4 has been added for NP-S; affiliations have been corrected from 1,2,3 to 3,4 for AN Search for more papers by this author Michal Mark-Daniei Michal Mark-Daniei Pediatric Stem Cell Research Institute, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Division of Pediatric Nephrology, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Search for more papers by this author Sara Pri-Chen Sara Pri-Chen Pediatric Stem Cell Research Institute, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Division of Pediatric Nephrology, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Search for more papers by this author Yehudit Gnatek Yehudit Gnatek Pediatric Stem Cell Research Institute, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Division of Pediatric Nephrology, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Search for more papers by this author Hadas Alfandary Hadas Alfandary Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Institute of Nephrology, Schneider Children's Medical Center of Israel, Petah Tikva, Israel Search for more papers by this author Nira Varda-Bloom Nira Varda-Bloom Division of Hematology and Cord Blood Bank, Sheba Medical Center, Ramat Gan, Israel Search for more papers by this author Dekel D Bar-Lev Dekel D Bar-Lev Pediatric Stem Cell Research Institute, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Division of Pediatric Nephrology, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Search for more papers by this author Naomi Bollag Naomi Bollag The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel Search for more papers by this author Rachel Shtainfeld Rachel Shtainfeld The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel Search for more papers by this author Leah Armon Leah Armon The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel Search for more papers by this author Achia Urbach Achia Urbach The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel Search for more papers by this author Tomer Kalisky Tomer Kalisky Faculty of Engineering, Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, Israel Search for more papers by this author Arnon Nagler Arnon Nagler Division of Hematology and Cord Blood Bank, Sheba Medical Center, Ramat Gan, Israel Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Correction added on 3 April 2017 after first online publication: affiliation 4 has been added for NP-S; affiliations have been corrected from 1,2,3 to 3,4 for AN Search for more papers by this author Orit Harari-Steinberg Orit Harari-Steinberg Pediatric Stem Cell Research Institute, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Division of Pediatric Nephrology, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Search for more papers by this author Jack L Arbiser Jack L Arbiser Department of Dermatology, Emory University School of Medicine, Atlanta, GA, USA Winship Cancer Institute, Atlanta Veterans Administration Hospital, Atlanta, GA, USA Search for more papers by this author Benjamin Dekel Corresponding Author Benjamin Dekel [email protected] [email protected] orcid.org/0000-0002-3784-8238 Pediatric Stem Cell Research Institute, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Division of Pediatric Nephrology, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Search for more papers by this author Author Information Oren Pleniceanu1,2,3,4, Racheli Shukrun1,2,4, Dorit Omer1,2, Einav Vax1,2,4, Itamar Kanter5, Klaudyna Dziedzic1,2,4, Naomi Pode-Shakked1,2,4, Michal Mark-Daniei1,2, Sara Pri-Chen1,2, Yehudit Gnatek1,2, Hadas Alfandary4,6, Nira Varda-Bloom3, Dekel D Bar-Lev1,2, Naomi Bollag7, Rachel Shtainfeld7, Leah Armon7, Achia Urbach7, Tomer Kalisky5, Arnon Nagler3,4, Orit Harari-Steinberg1,2, Jack L Arbiser8,9 and Benjamin Dekel *,*,1,2,4 1Pediatric Stem Cell Research Institute, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel 2Division of Pediatric Nephrology, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Ramat Gan, Israel 3Division of Hematology and Cord Blood Bank, Sheba Medical Center, Ramat Gan, Israel 4Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel 5Faculty of Engineering, Institute of Nanotechnology, Bar-Ilan University, Ramat Gan, Israel 6Institute of Nephrology, Schneider Children's Medical Center of Israel, Petah Tikva, Israel 7The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel 8Department of Dermatology, Emory University School of Medicine, Atlanta, GA, USA 9Winship Cancer Institute, Atlanta Veterans Administration Hospital, Atlanta, GA, USA *Corresponding author. Tel: +972 3 5302445; Fax: +972 3 5303637; E-mail: [email protected] or [email protected] EMBO Mol Med (2017)9:508-530https://doi.org/10.15252/emmm.201506111 Correction(s) for this article Peroxisome proliferator-activated receptor gamma (PPARγ) is central to the initiation and propagation of human angiomyolipoma, suggesting its potential as a therapeutic target01 December 2017 Correction added on 1 December 2017, after first online publication: the term “PPARG” has been modified to “PPARγ” in the title, abstract and keywords to improve searchability. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Angiomyolipoma (AML), the most common benign renal tumor, can result in severe morbidity from hemorrhage and renal failure. While mTORC1 activation is involved in its growth, mTORC1 inhibitors fail to eradicate AML, highlighting the need for new therapies. Moreover, the identity of the AML cell of origin is obscure. AML research, however, is hampered by the lack of in vivo models. Here, we establish a human AML-xenograft (Xn) model in mice, recapitulating AML at the histological and molecular levels. Microarray analysis demonstrated tumor growth in vivo to involve robust PPARγ-pathway activation. Similarly, immunostaining revealed strong PPARγ expression in human AML specimens. Accordingly, we demonstrate that while PPARγ agonism accelerates AML growth, PPARγ antagonism is inhibitory, strongly suppressing AML proliferation and tumor-initiating capacity, via a TGFB-mediated inhibition of PDGFB and CTGF. Finally, we show striking similarity between AML cell lines and mesenchymal stem cells (MSCs) in terms of antigen and gene expression and differentiation potential. Altogether, we establish the first in vivo human AML model, which provides evidence that AML may originate in a PPARγ-activated renal MSC lineage that is skewed toward adipocytes and smooth muscle and away from osteoblasts, and uncover PPARγ as a regulator of AML growth, which could serve as an attractive therapeutic target.2 Synopsis Angiomyolipoma (AML) research is hampered by the lack of an animal model. In a novel in vivo human AML model, PPARG inhibition is identified as a potential therapy and the AML cell of origin as a resident MSC/pericyte skewed toward adipogenic differentiation. An AML xenograft (Xn) model, exhibiting the classic histology, immunophenotype, and gene expression pattern of human AML, was established by injection of human AML cells in mice. AML Xn propagation in vivo is accompanied by robust activation of the PPARG pathway, and human AML tumors strongly express PPARG. PPARG is expressed across all tumor compartments, indicating that rather than accompanying adipocytic differentiation, PPARG activation drives AML growth, and with the presence of fat in the tumor being its by-product. PPARG antagonism, via small molecules and shRNA, results in strong growth inhibition of AML, as well as abrogation of tumor initiation in mice. Human AML cells exhibit striking similarity to human MSCs, in terms of gene expression, surface marker expression, and potential. Introduction Angiomyolipoma (AML) is the most common benign renal tumor and is characterized by a unique histology, consisting of blood vessels, smooth muscle, adipose tissue, and epithelioid cells in varying proportions (Folpe & Kwiatkowski, 2010). AML can develop both sporadically and as part of tuberous sclerosis complex (TSC) (Folpe & Kwiatkowski, 2010), an autosomal dominant disease characterized by the development of tumors in various organs. TSC significantly impacts patients' lives, mainly due to brain and kidney lesions. While the former represent the main cause of morbidity (e.g., seizures), the latter are the major cause of mortality. Renal lesions in TSC are diverse and include both epithelial (e.g., renal cysts) and mesenchymal lesions (AML). AML can be fatal when complicated by massive hemorrhage or renal failure (Crino et al, 2006). Moreover, an aggressive variant, termed “epithelioid” AML, has been described and shown to possess metastatic potential (Konosu-Fukaya et al, 2014). TSC is thought to result from loss of function of hamartin or tuberin (encoded by TSC1 and TSC2, respectively), two tumor suppressors, normally acting in a complex to inactivate mammalian target of rapamycin complex 1 (mTORC1). TSC1/2 inactivation results in enhanced mTORC1 activity, leading to unrestrained cell growth and proliferation. Hence, it is currently believed that TSC-related tumors arise in part due to mTORC1 activation (Kenerson et al, 2002). However, clinical findings suggest that additional signaling pathways contribute to their development. For instance, clinical trials in TSC patients have demonstrated that mTORC1 inhibitors fail to eradicate AML (Bissler et al, 2008, 2013). The uncovering of these additional pathways, however, has been hampered by the absence of an in vivo model of AML. Interestingly, TSC1/2-deficient animals develop various renal tumors, including renal cysts and carcinomas (both characteristic of TSC) but not AML (Kobayashi et al, 1995, 1999, 2001; Liang et al, 2014), underscoring the need for an animal model of human AML. In addition, this further supports the notion that dysregulation of the TSC1/2-mTORC1 pathway cannot fully explain AML growth. Although AML was initially considered a hamartoma, it was later shown to be a clonal lesion and thus a true neoplasm (Kattar et al, 1999), prompting the search for its cell of origin. However, the exact identity of the AML cell of origin has not yet been uncovered. Based on histopathology, AML is thought to derive from a perivascular epithelioid cell (PEC), a cell type whose normal counterpart is currently unknown. AML is therefore a member of the PEComa group of tumors, defined by the World Health Organization as mesenchymal tumors composed of histologically and immunohistochemically distinctive PECs (Bonetti et al, 1994). Importantly, the pathological diagnosis of AML requires co-expression of melanocytic (e.g., HMB-45) and muscle-related markers [e.g., αSMA (α smooth muscle actin)] (Folpe & Kwiatkowski, 2010). Notably, AML can also arise in extra-renal sites [e.g., liver (Goodman & Ishak, 1984), heart (Shimizu et al, 1994), and skin (Fitzpatrick et al, 1990)], indicating that its cell of origin is present throughout the body. Mesenchymal stem cells (MSCs), once considered the stem cells of mesenchymal tissues (Caplan, 2005), are currently perceived as a subpopulation of pericytes, residing in virtually every tissue, including the kidney (Crisan et al, 2008). We previously isolated and characterized an MSC-like cell type in the mouse kidney interstitium harboring broad mesenchymal potential (Dekel et al, 2006). Due to the various sources from which MSCs can be obtained, as well as their considerable heterogeneity, minimal criteria for their definition have been formulated (Dominici et al, 2006). These include plastic adherence, a typical antigenic profile and multipotency. In this report, we used serial xenografting of AML cells to establish an in vivo model of human AML, which recapitulated the biology of the tumor at the histological, immunohistochemical, and molecular levels. In order to uncover the mechanisms involved in AML growth, we interrogated gene expression along xenograft (Xn) propagation. Microarray gene expression analysis revealed strong activation of peroxisome proliferator-activated receptor gamma (PPARγ, henceforth referred to as PPARG1), a nuclear receptor and transcription regulator (Lehrke & Lazar, 2005) that is expressed in common epithelial tumors (e.g., breast and esophageal carcinoma) (Takahashi et al, 2006; Yuan et al, 2012). Immunohistochemical stainings (IHC) confirmed PPARG activation at the protein level both in AML-Xn and in primary human AML. Consequently, we show that PPARG inhibition significantly and specifically halts the in vitro growth of both sporadic and TSC-related AML cells and strongly limits their tumor-initiation capacity. We further demonstrate that PPARG inhibition leads to downregulation of the TGFB1 pathway, and specifically by inhibition of PDGFB and CTGF, two cardinal regulators of MSC/pericyte proliferation and function. Accordingly, we demonstrate that AML-Xn initiating cells fit within MSC criteria, thereby implicating a PPARG-activated resident renal MSC/pericyte lineage, which is therefore skewed toward the adipogenic and myogenic lineages, as the cell of origin of renal AML. Results Establishment of a human AML xenograft model in mice In order to gain insight into the mechanisms underlying AML development, we first wished to establish an in vivo model of human renal AML. For this purpose, we used two cell lines derived from two renal AML patients: “UMB”, derived from a TSC-related tumor and “SV7”, derived from a sporadic tumor (Arbiser et al, 2001). We subcutaneously injected 106 cells of each cell line into NOD/SCID mice. While UMB cells generated a slow-growing tumor approximately 4.5 months post-transplant, none of the mice injected with SV7 cells developed a tumor within 6 months, consistent with previous reports (Arbiser et al, 2001). Dissociation of the UMB-derived tumor into single cells and re-injection of 106 cells into secondary and then tertiary recipients resulted in tumor propagation and Xn formation. Upon serial injections, the interval necessary for tumor growth became increasingly and significantly shorter. Injection of first-generation Xn (T1)-derived cells resulted in the formation of a palpable tumor within 50 days ± 5 days, whereas third-generation Xn (T3)-derived cells generated tumors within 23 days ± 3 days (Fig 1A). Upon histological analysis, T1-Xn consisted mainly of densely growing atypical cells (Fig 1B). Although the tumor did not exhibit characteristic AML histology, scattered lipid-containing cells could be noted (Fig 1B). Histological analysis of T4-Xn revealed the presence of the three cellular components of classical AML: (i) blood vessels surrounded by PEC-like cells; (ii) “lipoid” areas, composed of large masses of atypical adipocytes; (iii) “myoid” areas, composed of spindle-shaped myoid cells (Fig 1B). These three components were surrounded by dense masses of undifferentiated small hyperchromatic cells. Of note, the same histology was established in all repetitions carried out (n = 3), using independent UMB cells (Appendix Fig S1). In addition, the morphological appearance was specific to AML-Xn, and not detected in any other Xn model established in our laboratory (Appendix Fig S2). So as to ascertain that the various cellular phenotypes seen in the Xn result from differentiation of human AML cells, we carried out IHC staining of the Xn for the human-specific marker HLA. Indeed, the vast majority of the tumor stained positive for HLA, including the lipoid and myoid areas (Fig 1C). Interestingly, examination of vessels in the tumor revealed that only perivascular cells, but not endothelial cells, were HLA+ (Fig 1D). IHC for the pericyte marker α-SMA confirmed that these HLA+ cells surrounding vessels are indeed pericytes (Fig 1D). Accordingly, immunofluorescent staining (IF) of T1- and T4-Xn using HLA and human-specific CD31 antibodies revealed only rare tubular structures lined by HLA+CD31+ cells in T4-Xn (Fig 1E) and no such structures in T1-Xn. These results support the notion that vessel formation in AML-Xn involves human AML cells assuming the role of pericytes, but not endothelial differentiation of tumor cells. In contrast, several vessels in the tumor were completely mouse-derived, as manifested by negative HLA staining in both endothelial and perivascular cells (Appendix Fig S3). In order to determine whether the Xn represent genuine human AML tumors, we next queried whether they recapitulate AML at the immunophenotypical level. To this end, we stained T1- and T4-Xn for HMB-45 and α-SMA, two diagnostic AML markers (Folpe & Kwiatkowski, 2010). HMB-45 demonstrated strong positive staining in both Xn generations (Fig 1F). In contrast, α-SMA was expressed only in T4-Xn (Fig 1F), demonstrating positive staining in both perivascular and non-perivascular cells. Finally, to validate the identity of the AML-Xn, we carried out IHC of the Xn for pS6, a marker of mTORC1 activation, a key feature of human AML. Both T1- and T4-Xn demonstrated pS6 expression, with the latter exhibiting robust and diffuse staining for pS6 (Fig 1G). Thus, propagation by cell transfer was successfully carried out, establishing a transmissible source of bona fide human AML tissue for further experimentation. Taken together, the triphasic histology and the distinctive “melano-myocytic” phenotype establish the Xn as a valid in vivo model of human AML. The ability to derive these Xn from UMB cells strongly suggests that the latter represent an equivalent of the tumor cell of origin. Notably, our results indicate that the characteristic vessels in AML do not result from endothelial differentiation of tumor cells. Rather, the latter seem to function as pericytes that recruit endothelial cells to form new vessels, in accordance with reports regarding the so-called PEC being the cell of origin of AML. In contrast, the other two lineages in AML (i.e., adipocytes and myocytes) seem to result from true differentiation of tumor cells. Figure 1. Characterization of AML xenografts (Xn) Growth interval between sequential Xn generations from 1st (T1) to 4th (T4), shown as mean ± SD (n ≥ 3); *P < 0.05; **P < 0.01 (one-way ANOVA with Bonferroni post hoc test). The exact P-values are specified in Appendix Table S5. T1-Xn harbor mainly undifferentiated cells with rare lipid-distended cells (arrows). In contrast, T4-Xn show the classic triphasic AML histology, consisting of vessels (left panel, arrow) surrounded by a thick layer of perivascular epithelioid cell (PEC)-like cells. Peripherally, a lipoid area, composed of lipid-distended cells, is seen. Furthermore, T4-Xn harbor “myoid” areas (right panel, arrow), composed of immature myocytes. Scale bar: 100 μm. Immunohistochemical staining (IHC) of T4-Xn for HLA, showing positive staining of the undifferentiated, lipoid, and myoid (left, middle, and right panels, respectively) compartments. Scale bar: 50 μm. IHC of T4-Xn for HLA (left panel) and pericyte marker α-SMA (right panel). Shown is a vessel, in which α-SMA+ pericytes are HLA+ (arrows), whereas endothelial cells are of mouse origin (arrowheads). Scale bar: 100 μm. Immunofluorescent staining (IF) of T4-Xn for HLA (green) and CD31 (red), demonstrating formation of human-derived vessels. Scale bar: 20 μm. IHC of T1- and T4-Xn for the diagnostic AML markers HMB-45 and α-SMA. HMB-45 is expressed in both T1- (left panel) and T4-Xn (middle panel) and stains both blood vessels and adipocytes. Scale bar: 200 μm. In contrast, only T4-Xn express α-SMA (right panel). Scale bar: 20 μm. IHC of T1- and T4-Xn for pS6, demonstrating expression in both T1- (left panel) and, to a greater extent, T4-Xn (middle panel). Scale bar: 50 μm. Note that within tumor vessels, only perivascular cells express pS6 (right panel). Scale bar: 20 μm. Download figure Download PowerPoint Molecular characterization of
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