LFG protein simulation
To visualize the 3D structure of the LFG protein, the sequence based SWISS-MODEL® software was used (Figure 1). Figure 1A shows the expected LFG monomer protein structure that consists of seven transmembrane domains (blue), that pass multiple times through the plasma membrane and are linked to a seperate protein tail (orange). Figure 1B shows the template alignment of LFG, as given by the uniprot protein data bank.
The high expression of LFG protein in osteosarcoma tissues
As above described, osteosarcoma samples were analyzed using commercially available immunofluorescence arrays. Table 1 gives an overview of the different anatomical localizations of the samples, the corresponding TNM classification, tumor grading, tumor stage, number of samples and mean LFG intensity. Osteosarcoma tissues from various anatomical regions were included, predominantly coming from femur and tibia (Table 1).
The mean LFG expression in osteosarcoma stage IIA (54,7; CI=47,1; 62,4) (p<0,05) was significantly higher than in normal bone marrow tissue (20,7; CI= 18,8; 22,8) (p<0,05). In stage IIB osteosarcoma, the mean LFG expression was calculated to 61,1 (CI=56,9; 65,3), being significantly higher compared to controls (p<0,05). Interestingly, the LFG expression seemed to correlate with the tumor stage, being higher in osteosarcoma stage IIB than in stage IIA as indicated by the relative fluorescent units (stage IIA 54,7 ± 7,7; stage IIB 61,1 ±4,2) (p<0,05) (Fig. 2, I). Samples with advanced osteosarcoma stage IIB had a larger tumor size, compared to IIA, and had the highest mean LFG expression of all osteosarcoma samples in the analysis. Both samples with osteosarcoma stage IIA and IIB had grade 3 tumors in which the sarcoma cells grew more aggressively and showed abnormal cell morphology. Figure 2, II shows a representative selection of LFG antibody stained fluorescent images of osteosarcomas. The increase in the LFG intensity in osteosarcoma stage IIB (Figure 2, IIC) , compared to stage IIA (Figure 2, IIB) and the controls (Figure 2, IIA) can be seen in the constant increase of green coloring from Figure 2, IIA to Figure 2, IIC.
The high expression of LFG protein in chondrosarcoma
Table 2 gives an overview of the different tumor localizations, tumor grading, tumor stage and mean LFG intensity of chondrosarcoma samples. The mean LFG expression in chondrosarcoma stage IA was significantly higher compared to normal bone marrow tissue (stage IA=44,3; controls=20,7) with a p-value <0,05 (Fig. 3,I). A significant higher LFG expression in chondrosarcoma stage IIA was oberserved (53,4; CI=42,4; 64,5) (p<0,05) compared to chondrosarcoma stage IA (36,1; CI=32,4; 39,8) (p<0,05) (Fig. 3,I). Although a significant increase in LFG expression could not be seen in all stages, the data assume a constant positive correlation between LFG expression and chondrosarcoma stages. The LFG expression level detected in stage IIB chondrosarcoma (49,3; CI=39,5; 59,1) was lower than in stage IIA (53,4; CI=42,4; 64,5) (p>0,05). Figure 3,II,A-E shows fluorescent images of chondrosarcoma samples with different tumor stages. The LFG intensity is expressed by green immunofluorescence. Healthy bone marrow tissue showed the lowest LFG expression compared to all subtypes of chondrosarcoma (Figure 3, IIA). The LFG expression increases from chondrosarcoma stage IA (Figure 3, IIB) to stage IB (Figure 3, IIC), as shown by an increase in intensitiy of the green immunoflourescence. Figure 2, IID shows the high LFG expression in chondrosarcoma stage IIA, significantly higher compared to controls (Figure 3, IIA) and stage IA chondrosarcoma (Figure 3, IIB). Figure 3, IIE shows the LFG expression of chondrosarcoma stage IIB being significantly higher compared to controls (Figure 3, IIA), but lower compared to stage IIA (Figure 3, IID).
The high expression of LFG protein in soft tissue sarcoma
Taking into consideration that the anti-apoptotic protein LFG showed high expression levels in osteosarcoma and chondrosarcoma, the expression of LFG in different subtypes of soft tissue sarcoma was analyzed. Table 3 shows the different soft tissue sarcoma subtypes that were investigated regarding LFG expression. A total number of 55 soft tissue sarcoma tissue samples were analyzed, using 5 skeletal muscle samples and 4 endometrium smooth muscle samples as unmatched controls. Table 3 gives an overview of the different soft tissue subtypes, the corresponding TNM classification, tumor grading, tumor stage and mean LFG intensity of the samples. The means of LFG expression were calculated for each soft tissue subtype as a whole by summarizing the data of all corresponding stages for each subtype (Table 3). Overall, the LFG expression variated strongly dependening on the soft tissue subtype (Figure 4A). Liposarcoma showed low LFG expression (47,4 ±4,2) (p<0,05), not noticeable compared to skeletal muscle control (45,8 ±3,9) (p<0,05), whereas malignant schwannoma (86,3 ±28,5) (p<0,05), dermatofibrosarcoma (65,9 ±9) (p<0,05) and leiomyosarcoma (90,6 ±25,4) (p<0,05) had a significantly higher LFG expression compared to skeletal muscle control (45,8 ±3,9) (p<0,05) and smooth muscle control (49,3 ±5,8) (p<0,05) (Figure 4A). The LFG expression in angiosarcoma (60,6 ±5,9) and synovial cell sarcoma (59,9) was higher compared to controls with the results not being significant (p>0,05) (Figure 4A). Figure 4B shows that LFG expression in dermatofibrosarcoma stage IA (63,5 ±8,8) (p<0,05) that was significantly higher compared to the controls (p<0,05), with an increase in LFG expression from stage IA (63,5 ±8,8) (p<0,05) to more advanced stage IB (82,3; no CI). The LFG expression correlated with malignant schwannoma tumor stage, increasing from stage IA (56,2; no CI) to stage IIA (74,7; no CI) and stage IIB (124,9; no CI) (Figure 4C). The LFG expression of malignant schwannoma stage III (89,6; no CI) was observed lower compared to stage IIB (124,9; no CI) (Figure 4C). The highest LFG expression of all specimen was detected in leiomyosarcoma stage IA (139,3; no CI) (Figure 4D). The LFG expression in leiomysarcoma stage IIB (77,0 ±15,4) (p<0,05) showed to be significantly higher compared to both controls (p<0,05) (Figure 4D).The LFG expression in leiomyosarcoma is exemplarily shown in Figure 5. Figure 5A shows an immunofluorescent image of endometrium smooth muscle as control with low LFG expression. The highest LFG expression of all specimen was detected in leiomyosarcoma stage IA presenting with multiple, widely distributed fluorescent signals (Figure 5B). Figure 5C shows the high LFG expression of leiomyosarcoma stage IIB, being significantly higher (p<0,05) compared to endometrium smooth muscle control (Figure 5A). Figure 5D shows an image of the high LFG expression that was observed in leiomyosarcoma stage III.