Heterogeneity and immunophenotypic plasticity of malignant cells in human liposarcomas

Abstract

Liposarcomas are tumors arising in white adipose tissue (WAT) with avidity for local recurrence. Aggressive dedifferentiated liposarcomas (DDLS) may arise from well-differentiated subtypes (WDLS) upon disease progression, however, this key issue is unresolved due in large part to knowledge gaps about liposarcoma cellular composition. Here, we wished to improve insights into liposarcoma cellular hierarchy. Tumor section analysis indicated that the populations, distinguishable based on the expression of CD34 (a marker of adipocyte progenitors) and CD36 (a marker of adipocyte differentiation), occupy distinct intra-tumoral locations in both WDLS and DDLS. Taking advantage of these markers, we separated cells from a panel of fresh human surgical specimens by fluorescence-activated cell sorting (FACS). Based on chromosome analysis and the culture phenotypes of the composing populations, we demonstrate that malignant cells comprise four mesenchymal populations distinguished by the expression of CD34 and CD36, while vascular (CD31+) and hematopoietic (CD45+) components are non-neoplastic. Finally, we show that mouse xenografts are derivable from both CD36-negative and CD36-positive DDLS cells, and that each population recreates the heterogeneity of CD36 expression in vivo. Combined, our results show that malignant cells in WDLS and DDLS can be classified according to distinct stages of adipogenesis and indicate immunophenotypic plasticity of malignant liposarcoma cells.

Figure 1

WDLS and DDLS cells at distinct differentiation stages. (A) A schematic depicting expression of CD34 and CD36, cell surface markers used for liposarcoma cell classification, during differentiation of mesenchymal adipocyte progenitors. (B) Immunolocalization of distinct liposarcoma populations in serial paraffin sections of representative WDLS and DDLS samples subjected to immunofluorescence with antibodies against CD36 or CD34 (red). CD36+CD34- cells with adipocyte morphology (*), adjacent stromal CD36+CD34+ cells (arrowheads) and mainly perivascular CD36-CD34+ cells (arrows) are indicated relative to non-malignant vasculature expressing CD31 (green). Nuclei are blue. Scale bar: 50 μm.

Figure 2

Characterization of WDLS and DDLS populations. (A) FACS separation of cells based on the expression of CD36, CD34, and CD45 in a representative WDLS. Percentages of cells positive for a marker among viable cells are indicated. (B) Identification of malignant cells through the detection of the 12q15 amplification (red) by FISH in CD36+ and CD34+ cells. (C) Morphology of cells sorted based on the expression of CD36, CD34, or CD45 from the WDLS tumor upon adherence. In (B-C), WDLS cells isolated in (A) were used. (D) FACS 3D contour graphs for representative samples of benign WAT, as well as for WDLS and DDLS samples analyzed in Fig. 1 (B). Gates for CD34dCD36d, CD34dCD36b, CD34bCD36-, and CD34bCD36+ cells are shown. (E) Percentages among viable cells analyzed in multiple samples were calculated for normal WAT, WDLS and DDLS (N=5 for each). Mean frequencies of each population +/- SD among viable tumor cells in are shown. * P<0.05. (F) Morphology of cells sorted in (D) from a representative DDLS sample upon plastic adherence in culture. FISH detecting the 12q15 amplicon (red) confirms cells in the four populations as malignant. Green: centromeric probe; blue: DAPI. Scale bar: 50 μm.

Figure 3

Culture plasticity and tumorogenicity of liposarcoma cells. (A) Separation of early passage Lipo863 cells derived from a representative DDLS sample (Fig. 2D) based on CD36 and CD34 expression (left) and morphology of sorted CD36+ and CD36- Lipo863 cells upon adherence (right). (B) Separation of cells derived from a Lipo863 mouse xenograft based on CD36 and CD34 expression (left) and morphology of CD36+ and CD36- cells sorted from the mouse Lipo863 tumor upon adherence (right). (C) Paraffin sections of xenografts grown from Lipo863 cells subjected to immunofluorescence with antibodies against CD36 or CD34 (red) and against CD31 (green). Arrows indicate CD36 expression. Nuclei are blue. Scale bar: 50 μm. (D) Tumor growth in mice xenografted with 10⁶ cells of the indicated Lipo863 (B) populations or with 10⁶ tumor-infiltrating mouse leukocytes. (E) Photographs of indicated tumor-bearing mice and resected tumors at week 12 post-grafting.

Figure 4

Plasticity of liposarcoma populations in mouse xenografts. (A) Separation of cells from secondary xenografts of CD36+ (left) and CD36- (right) cells (Figure 3D-E) into CD36+ and CD36- populations. (B) Paraffin sections of xenografts grown from CD36+ cells (left) and CD36- cells (right) subjected to immunofluorescence with antibodies against CD36 (red) indicating comparable distribution of CD36-expressing cells relative to vasculature expressing CD31 (green). Nuclei are blue. Scale bar: 50 μm. (C) A model of liposarcomagenesis. Liposarcoma arises in benign WAT as a result of 12q13-15 amplification and MDM2 overexpression (red nucleus), which occurs at one of the stages of adipogenesis (box). DDLS arise either from WDLS though gradual dedifferentiation of progressive percentage of tumor cells to the ASC-like phenotype or independently of WDLS by separate mutations in WAT leading to a comparatively more aggressive domination of malignant ASC-like cells.