Additional file 1 of Architecting the metabolic reprogramming survival risk framework in LUAD through single-cell landscape analysis: three-stage ensemble learning with genetic algorithm optimization

Supplementary Material

Tracking the fate of cancer-stromal hybrids.

<p>Representative morphologies of the hybrid cells during colony formation are shown. <b>A</b>, Two weeks into the culture, most of the hybrids contained two nuclei of similar fluorescence. No cell division was observed. <b>B</b>, Four weeks into the co-culture, hybrid cells adopted atypical morphology with multiple nuclei. No cell division was observed. <b>C</b>, Six weeks into the culture, the remaining hybrid cells became thin or narrow, with multiple nuclei in segments of the cell. <b>D</b>, Eight weeks into the culture, cell division became prevalent. The cell division was abnormal because it produced daughter cells in varied shapes and with reduced viability. For each view, a phase contrast image (top) and red fluorescence image (bottom) are shown. When necessary, arrows are used to indicate nuclei. All the images are shown at 200× magnification.</p

Confirming cancer-stromal cell fusion.

<p>Representative cancer-stromal cell fusion events from the co-culture of RL-1 cells with hMSC-GFP cells are shown. <b>A</b>, A single fusion event at day 7 is shown in bright field, green fluorescence, and red fluorescence. The green and red fluorescence images are merged (merged fluorescence) to show the two nuclei of different fluorescence. <b>B</b>, Merged fluorescence images for 4 additional fusion events are shown, with events 1 and 2 recorded at day 7, and 3 and 4 at day 14. Arrows are used to indicate nuclei. All the images are shown at 200× magnification.</p

Characteristics of the spontaneous cancer-stromal cell fusion.

<p>RL-1 cells (<b>A</b>) and HPS-15 cells (<b>B</b>) are shown in separate culture. After 7 days of co-culture, spontaneous fusion could be seen (<b>C</b>). At higher magnification, the fused cell contained two nuclei, one fluorescently red and the other fluorescently pale (<b>D</b>). Cancer-stromal fusion was frequently seen in areas where RL-1 and HPS-15 formed close contact (<b>E</b>). In some cases, cells in the middle of a fusion could be seen (<b>F</b>). The two nuclei could be seen close to each other (<b>G</b>) or separated (<b>H</b>). For each view, a phase contrast image (top) and red fluorescence image (bottom) are shown. Arrows are used to indicate nuclei.</p

Time-dependence of cancer-stromal cell fusion.

<p>Co-cultures of RL-1 and HPS-15 cells were observed weekly for frequency of cell fusion. For each view, a phase contrast image (top) and red fluorescence image (bottom) are shown. Arrows are used to indicate cancer-stromal cell fusion events. All the images are shown at 40× magnification.</p

Genotypic and phenotypic changes in the derivative clones from cancer-stromal fusion.

<p>Genotypic and phenotypic parameters of the first 9 clones of the RL-1 and HPS-15 fusion hybrids were compared to those of the first 12 clones from control cloning. Compared to RL-1 clones, all the derivative clones lost Y chromosomes (<b>A</b> versus <b>B</b>). Detected by Western blotting, some of the derivative clones showed persistent AR expression even under androgen-deprivaton (<b>C</b> versus <b>D</b>). In these studies, cells were cultured for 48 hours in regular culture medium (C), androgen deprivation medium (−), and androgen deprivation medium containing 5 nM R1881 (+). The derivative clones were detected to express increased levels of PSA, even during androgen-deprivation (<b>E</b> versus <b>F</b>). When growth rate was assayed by MTT conversion, clones derived from cancer-stromal fusion displayed accelerated growth in androgen-independent fashion (<b>G</b> versus <b>H</b>). Data represent the mean of triplicate assays. For all the data points, standard deviation was less than 5% of the mean and is not shown.</p

Reduced colony formation in cancer-stromal fusion hybrids.

a<p>Wells containing a single cell 24 hours after the plating were enumerated.</p>b<p>Colonies from the wells containing a single cell were enumerated.</p>c<p>Data were from one colony formation assay.</p>d<p>Data were combined results from 4 repeated colony formation assays.</p

OA impairs the HDAC4 nuclear import induced by CTS.

<p>(A) Fluorescence microscope showed GFP-HDAC4 was mainly located in the nucleus in cells subjected to CTS only (a-c), and mainly in the cytoplasm in cells subjected to CTS with OA (d-f). Green indicated the GFP-HDAC4, and blue indicated cell nucleus stained by Hoechst 33342. (B) Percentage of green GFP-HDAC4 located only in nucleus was scored. 300 cells from 3 independent experiments were counted. Data were expressed as means±SD (P = 0.02). (C) Nuclear and cytoplasmic lysates were separated and followed by western blot analysis with anti-HDAC4 antibody. Histone 3 and GAPDH acted as loading controls for the nuclear and cytoplasmic fraction respectively (C-a). Semi-quantitative assay of band densities showed that cytoplasmic HDAC4 was decreased, and nuclear HDAC4 was increased in CTS group as compared to control group and CTS with OA group; The relative grey value of HDAC4 had no statistics difference between control group and CTS with OA group in both cytoplasm and nucleus (C-b). Values were presented as mean±SD (n = 3), *P<0.05.</p

Overview of experimental design.

<p>(A) Workflow scheme for analysis of the effect of cyclic equibiaxial tensile strain (CTS) on HDAC4 relocation in chondrocytes. After isolated and then cultured for 6 days, the passage chondrocytes were transfected with GFP-HDAC4. At 48 h post-transfection, the transfected cells were submitted to CTS for 3 hours. (B) The chondrocytes at Area A were subjected to 6% equibiaxial CTS. (C) Green fluorescent protein (GFP) was captured by fluorescence microscope to validate the transfection efficiency of GFP-HDAC4 in chondrocytes. Nuclei were stained by Hoechst 33342 (blue) (C-a). 300 cells from 3 independent experiments were counted. Transfection efficiency of GFP-HDAC4 is 96%±3.64% (C-b). (D, E) The time-dependent changes in gene expression levels for aggrecan and type II collagen following CTS. Real-time PCR showed that mRNA level for aggrecan (D) and collagen II (E) were elevated after 2 and 3 h of CTS, but decreased after 4 h of CTS. Values are presented as mean±SD (n = 3). *P<0.05, **P<0.01 versus the non-stretched control group.</p

Morphologic changes in the derivative clones during colony formation.

<p>The morphology of a derivative colony was followed during the cloning process, growing from a single well of a 96-well plate to a single well of 24-well plate, to a single well of a 6-well plate, and to a 10 cm dish. For each view, a phase contrast image (top) and red fluorescence image (bottom) are shown. All the images are shown at 100× magnification.</p