Limits...
Defining Clonal Color in Fluorescent Multi-Clonal Tracking.

Wu JW, Turcotte R, Alt C, Runnels JM, Tsao H, Lin CP - Sci Rep (2016)

Bottom Line: Combinatorial fluorescent protein expression in germline cells has proven its utility for tracking the formation and regeneration of different organ systems.However, the assignment of clonal identity requires an analytical framework in which clonal markings can be parameterized and validated.We then demonstrate refined clonal trackability of melanoma cells using this scheme.

View Article: PubMed Central - PubMed

Affiliation: Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.

ABSTRACT
Clonal heterogeneity and selection underpin many biological processes including development and tumor progression. Combinatorial fluorescent protein expression in germline cells has proven its utility for tracking the formation and regeneration of different organ systems. Such cell populations encoded by combinatorial fluorescent proteins are also attractive tools for understanding clonal expansion and clonal competition in cancer. However, the assignment of clonal identity requires an analytical framework in which clonal markings can be parameterized and validated. Here we present a systematic and quantitative method for RGB analysis of fluorescent melanoma cancer clones. We then demonstrate refined clonal trackability of melanoma cells using this scheme.

No MeSH data available.


Related in: MedlinePlus

Clonal founder cell selection, clonal chromatic mode and chromatic spread.(a) Combinatorial gating scheme for single clonal founder cell sorting. RGB channels were gated by intensity and different combinations of these R/G/B channel gates were used to select single cells from MelaChromas for clonal expansion. MelaChromas of various MOIs (0.7, 2.8, 4.9) were used the source of founder cells. As a result, a wide variety of FP expression levels and Cerulean(B):Venus(G):tdTomato(R) ratios were represented in the selected cells, as shown in the confocal images of expanded clones. For visual reference, confocal images of 1FP-expressing clones and un-transduced A375 cells (A375-WT) at 50% magnification are also shown. Scale bar: 25 μm. (b) Spherical scatter plots of the clones in (a). 1E4 cells were analyzed by flow cytometry per clone. (c) Spherical histogram of clone #2 in (a,b). (d) Chromatic mode (crosshair) and chromatic spreads (pink) of clone #2. Chromatic spreads, as projections of isosurfaces drawn at 50%, 25%, 10%, 5%, 2%, and 1% of the highest cell count at the chromatic mode (Supplementary Fig. S4), co-localized with the high cell count region in the clonal spherical histogram (same as (c), shown magnified in white). Values indicate the actual percentage of clonal cells with chromaticity values enclosed within each contour. (e), Spherical scatter plot showing the chromatic mode coordinates of the 256 expanded MelaChroma clones.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4829845&req=5

f2: Clonal founder cell selection, clonal chromatic mode and chromatic spread.(a) Combinatorial gating scheme for single clonal founder cell sorting. RGB channels were gated by intensity and different combinations of these R/G/B channel gates were used to select single cells from MelaChromas for clonal expansion. MelaChromas of various MOIs (0.7, 2.8, 4.9) were used the source of founder cells. As a result, a wide variety of FP expression levels and Cerulean(B):Venus(G):tdTomato(R) ratios were represented in the selected cells, as shown in the confocal images of expanded clones. For visual reference, confocal images of 1FP-expressing clones and un-transduced A375 cells (A375-WT) at 50% magnification are also shown. Scale bar: 25 μm. (b) Spherical scatter plots of the clones in (a). 1E4 cells were analyzed by flow cytometry per clone. (c) Spherical histogram of clone #2 in (a,b). (d) Chromatic mode (crosshair) and chromatic spreads (pink) of clone #2. Chromatic spreads, as projections of isosurfaces drawn at 50%, 25%, 10%, 5%, 2%, and 1% of the highest cell count at the chromatic mode (Supplementary Fig. S4), co-localized with the high cell count region in the clonal spherical histogram (same as (c), shown magnified in white). Values indicate the actual percentage of clonal cells with chromaticity values enclosed within each contour. (e), Spherical scatter plot showing the chromatic mode coordinates of the 256 expanded MelaChroma clones.

Mentions: We devised a strategy to isolate chromatically diverse founder cells for clonal expansion. Our goal was to build a library of diversely color-coded clones with representative clonal color properties, which would also serve as potential participant clones in a multi-clone tracking study. In vitro cultures of individual clones and flow cytometry allow repeatable clonal color data collection at high cell counts per clone. We used MelaChroma cells of MOIs 0.7, 2.8 and 4.9 as our source of founder cells. These MelaChromas had different distributions of intracellular FP concentrations, corresponding to different RGB intensities. To avoid oversampling of high frequency chromaticities in each MelaChroma, we created a combinatorial FACS gating scheme for founder cell sorting that further ensured selection of cells with diverse RGB ratios (Fig. 2a,b).


Defining Clonal Color in Fluorescent Multi-Clonal Tracking.

Wu JW, Turcotte R, Alt C, Runnels JM, Tsao H, Lin CP - Sci Rep (2016)

Clonal founder cell selection, clonal chromatic mode and chromatic spread.(a) Combinatorial gating scheme for single clonal founder cell sorting. RGB channels were gated by intensity and different combinations of these R/G/B channel gates were used to select single cells from MelaChromas for clonal expansion. MelaChromas of various MOIs (0.7, 2.8, 4.9) were used the source of founder cells. As a result, a wide variety of FP expression levels and Cerulean(B):Venus(G):tdTomato(R) ratios were represented in the selected cells, as shown in the confocal images of expanded clones. For visual reference, confocal images of 1FP-expressing clones and un-transduced A375 cells (A375-WT) at 50% magnification are also shown. Scale bar: 25 μm. (b) Spherical scatter plots of the clones in (a). 1E4 cells were analyzed by flow cytometry per clone. (c) Spherical histogram of clone #2 in (a,b). (d) Chromatic mode (crosshair) and chromatic spreads (pink) of clone #2. Chromatic spreads, as projections of isosurfaces drawn at 50%, 25%, 10%, 5%, 2%, and 1% of the highest cell count at the chromatic mode (Supplementary Fig. S4), co-localized with the high cell count region in the clonal spherical histogram (same as (c), shown magnified in white). Values indicate the actual percentage of clonal cells with chromaticity values enclosed within each contour. (e), Spherical scatter plot showing the chromatic mode coordinates of the 256 expanded MelaChroma clones.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4829845&req=5

f2: Clonal founder cell selection, clonal chromatic mode and chromatic spread.(a) Combinatorial gating scheme for single clonal founder cell sorting. RGB channels were gated by intensity and different combinations of these R/G/B channel gates were used to select single cells from MelaChromas for clonal expansion. MelaChromas of various MOIs (0.7, 2.8, 4.9) were used the source of founder cells. As a result, a wide variety of FP expression levels and Cerulean(B):Venus(G):tdTomato(R) ratios were represented in the selected cells, as shown in the confocal images of expanded clones. For visual reference, confocal images of 1FP-expressing clones and un-transduced A375 cells (A375-WT) at 50% magnification are also shown. Scale bar: 25 μm. (b) Spherical scatter plots of the clones in (a). 1E4 cells were analyzed by flow cytometry per clone. (c) Spherical histogram of clone #2 in (a,b). (d) Chromatic mode (crosshair) and chromatic spreads (pink) of clone #2. Chromatic spreads, as projections of isosurfaces drawn at 50%, 25%, 10%, 5%, 2%, and 1% of the highest cell count at the chromatic mode (Supplementary Fig. S4), co-localized with the high cell count region in the clonal spherical histogram (same as (c), shown magnified in white). Values indicate the actual percentage of clonal cells with chromaticity values enclosed within each contour. (e), Spherical scatter plot showing the chromatic mode coordinates of the 256 expanded MelaChroma clones.
Mentions: We devised a strategy to isolate chromatically diverse founder cells for clonal expansion. Our goal was to build a library of diversely color-coded clones with representative clonal color properties, which would also serve as potential participant clones in a multi-clone tracking study. In vitro cultures of individual clones and flow cytometry allow repeatable clonal color data collection at high cell counts per clone. We used MelaChroma cells of MOIs 0.7, 2.8 and 4.9 as our source of founder cells. These MelaChromas had different distributions of intracellular FP concentrations, corresponding to different RGB intensities. To avoid oversampling of high frequency chromaticities in each MelaChroma, we created a combinatorial FACS gating scheme for founder cell sorting that further ensured selection of cells with diverse RGB ratios (Fig. 2a,b).

Bottom Line: Combinatorial fluorescent protein expression in germline cells has proven its utility for tracking the formation and regeneration of different organ systems.However, the assignment of clonal identity requires an analytical framework in which clonal markings can be parameterized and validated.We then demonstrate refined clonal trackability of melanoma cells using this scheme.

View Article: PubMed Central - PubMed

Affiliation: Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.

ABSTRACT
Clonal heterogeneity and selection underpin many biological processes including development and tumor progression. Combinatorial fluorescent protein expression in germline cells has proven its utility for tracking the formation and regeneration of different organ systems. Such cell populations encoded by combinatorial fluorescent proteins are also attractive tools for understanding clonal expansion and clonal competition in cancer. However, the assignment of clonal identity requires an analytical framework in which clonal markings can be parameterized and validated. Here we present a systematic and quantitative method for RGB analysis of fluorescent melanoma cancer clones. We then demonstrate refined clonal trackability of melanoma cells using this scheme.

No MeSH data available.


Related in: MedlinePlus