PPARG is central to the initiation and propagation of human angiomyolipoma, suggesting its potential as a therapeutic target

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 PPARG‐pathway activation. Similarly, immunostaining revealed strong PPARG expression in human AML specimens. Accordingly, we demonstrate that while PPARG agonism accelerates AML growth, PPARG 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 PPARG‐activated renal MSC lineage that is skewed toward adipocytes and smooth muscle and away from osteoblasts, and uncover PPARG as a regulator of AML growth, which could serve as an attractive therapeutic target.


Supplementary methods
Hematoxylin and eosin (H&E) staining 5 µm sections of paraffin-embedded Xn tissues were mounted on super frost/plus glass and incubated at 60°`C for 40 minutes. After de-paraffinization, slides were incubated in Mayer's Hematoxylin solution (Sigma-Aldrich) and incubated with 1% Hydrochloric acid in 70% ethanol for 1 minute. Slides were then incubated for 10 seconds in Eosin (Sigma-Aldrich). Images were produced using Olympus BX51TF fluorescent microscope with Olympus DP72 camera and cellSens standard software.

Immunohistochemical (IHC) and immunofluorescent (IF) staining of paraffin-embedded Xn tissues
Sections were pre--treated using OmniPrep solution (Zytomed Systems) in 95°C for 1 hour according to the manufacturer's protocol. For IHC, blocking was performed using Cas--Block solution (Invitrogen) for 20 minutes followed by 1 hour of incubation at room temperature (RT) with primary antibodies for HLA, α-SMA, CD31, HMB-45, PPARγ and pS6 (Appendix Table   S6). Samples were incubated with secondary antibodies (ImmPRESS TM anti mouse/rabbit reagent peroxidase, Vector labs) for 30 minutes at RT and detected using ImmPACT DAB kit (Vector) according to the manufacturer's protocol. Hematoxylin was used for counterstaining. For IF, sections were blocked with Cas--Block solution (Invitrogen) for 1 hour followed by incubation with primary antibodies for HLA and CD31 (Appendix Table S6) and then washed and incubated with secondary antibodies (Appendix Table S7) for 1 hour. Mounting containing DAPI (Southern Biotech) was applied. Photos were obtained using Olympus BX51TF fluorescent microscope with Olympus DP72 camera and cellSens standard software.

IF staining of cells
Cells were grown on coverslips in 24-well culture dishes overnight, after which they were washed with PBS, fixed in 3% paraformaldehyde (PFA) for 10 minutes at RT and washed with PBS 3 times for 5 minutes at RT. The cells were next permeabilized for 5 minutes with 0.2% (vol/vol) Triton X-100 in PBS at RT, washed 3 times with PBST (PBS with 0.05% Tween-20) for 5 minutes and then blocked with Cas--Block solution (Invitrogen) for 1 hour at RT. Next, the cells were incubated with the primary antibody (PPARg, Santa-Cruz, and β-Actin, Cell signaling) for 1 hour at RT, washed 3 times with PBST, incubated with the secondary antibody (as described above) for 1 hour at RT and washed three times with PBST. Finally, mounting with DAPI (Southern Biotech) was applied. Photos were obtained using Olympus BX51TF fluorescent microscope with Olympus DP72 camera and cellSens standard software.

Flow Cytometry
10 5 Cells were suspended in FACS buffer (0.5% BSA, 2 mM EDTA in 1× PBS) and blocked with FcR blocking reagent (MiltenyiBiotec) and human serum (1:1). The cells were then incubated with a primary antibody or an isotype control (Appendix Table S8). Cells were incubated with a secondary antibody if needed (Appendix Table S8). Cell viability was tested using 7AAD viability staining solution (eBioscience). Cell labelling was detected using FACSCalibur (BD Pharmingen). FACS results were analyzed using FlowJo analysis software.
For flow cytometry analysis of AML Xn, freshly removed Xn were dissociated into a single cell suspension as described above and immediately analyzed.

Quantitative real--time RT--PCR analysis of gene expression
Total RNA was isolated using TRIzol reagent (Life Technologies) according to the manufacturer's instructions. cDNA was synthesized using the High Capacity cDNA reverse transcription kit (Applied Biosystems, ABI). Quantitative Real--time RT--PCR (qRT--PCR) was performed using StepOnePlus Real-Time PCR System (ABI) and the specific TaqMan Gene Expression assays for the relevant genes in the presence of TaqMan Fast Universal PCR Master Mix (both from ABI). hHPRT1, hGAPDH and hB2M were used as endogenous controls. The results were analyzed using StepOnePlus Real-time Software in the ΔΔCT method which determines fold change in gene expression relative to a comparator sample. Statistical analysis was performed using a non--paired 2--tailed t--test. Statistical significance was considered to be p-value <0.05.

Human FK and AK tissue
Normal human 16 week gestation kidney was obtained following curettage of elective abortions.
Normal human AK samples were retrieved from borders of renal cell carcinoma tumors from patients who underwent partial nephrectomy. Fetal and adult kidney tissues were handled within 1 h following the curettage or nephrectomy procedures, respectively. Collected tissues were washed with cold Hank's Balanced Salt Solution (HBSS) (Invitrogen, Carlsbad, Calif., USA) and cut into 0.5 cm cubes by sterile surgical scalpels. The pieces were then used for total RNA extraction with TRIzol (Life Technologies, Invitrogen, Carlsbad, Calif., USA).

Cell Culture
AML cell lines and Xn cells were grown in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 1% Penicillin-streptomycin 100M and 1% L--glutamine (both from Biological Industries). Human AK and FK and WT cells were grown as previously described

Supplementary figures
Appendix Figure  S1 Appendix Figure S1: Validation of the human AML-Xn model: Shown are two representative repetitions of the newly established Xn model, entitled UMB2-Xn and UMB3-Xn. Shown is the histology of the 5 th (T5) generation of Xn in mice. Both UMB2-T5 (A) and UMB3-T5 (B) demonstrate in H&E staining, the same histological features consistent with the parental tumor, namely the presence of blood vessels (black arrows), adipocytes (arrowheads) and spindle-shaped, early myoid cells (white arrow). In addition, these tumors as well demonstrate strong nuclear expression of PPARG (C&D). Notably, PPARG expression PPARG is robustly expressed in the mass of undifferentiated cells of the tumors (C, insert). In addition, PPARG is expressed in both adipocytic and myoid cells within the tumors (D, arrow). Scale bar: 100 µm (except for insert, 50 µm).
Appendix Figure S2: H&E staining of Wilms' tumor (WT) and Pleuro-Pulmonary Blastoma: In order to demonstrate the specificity of the human AML-Xn model, we have characterized Xn tumors of two different tumors: WT and PPB, representing a renal and non-renal tumors. Notably, both Xn models exhibit a significantly different histological appearance compared to the AML-Xn model, which is at the same time reminiscent of the parental neoplasm. WT-Xn (left and middle panels) demonstrate the classical tri-phasic histology, consisting of renal tubules (T), dense blastema (B) and stroma (S). In contrast, PPB-Xn (right panel) exhibit the typical hyper-cellular primitive blastemal-like morphology.
Appendix Figure S9: Ingenuity pathway analysis (IPA) of differentially-expressed genes leading to the decreased vasculogenesis cell survival seen in AML cells treated for 24h with GW9662 compared with control, vehicle-treated cells. Genes in green were down-regulated while genes in red were up-regulated, with darker colors indicating a more significant change.
Appendix Figure S10: Representative low magnification photograph of T5-Xn, demonstrating the general composition of the Xn. Most of the tumor is seen to consist of dense masses of un-differentiated small hyperchromatic cells, alongside blood vessels, adipocytes and myoid areas.

Supplementary tables
Appendix  Table S1: Ingenuity pathway analysis (IPA) of PPARG-dependent genes demonstrating significant change between T1-Xn and T5-Xn according to cellular functions.