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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

ct-MelaChroma’s clonal composition over time.(a) Mean frequency of participant clones of ct-MelaChroma on various days post pooling. Emergence dominance of three clones (01, 02, 03) was evident by Day 8. (b) Clonal composition of ct-MelaChroma over the course of fifteen weeks. The 12% clonal frequency cutoff at Week 2 separated the clones that trended towards dominance (clones 01–3) versus withdrawal (clones 04–15). Error bars denote standard deviations of the six samples analyzed by flow cytometry for the day except for Days 51 and 72, on which two samples were analyzed. 1.35–2.32E6 cells were clonally assigned, per sample, on all days. (c) Mono-clonal growth curves of five of ct-MelaChroma’s participant clones. WT denotes un-transduced A375 cells. Four measurements were made per time point between two consecutive sub-cultures. Error bars indicate standard errors for each measurement.
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f4: ct-MelaChroma’s clonal composition over time.(a) Mean frequency of participant clones of ct-MelaChroma on various days post pooling. Emergence dominance of three clones (01, 02, 03) was evident by Day 8. (b) Clonal composition of ct-MelaChroma over the course of fifteen weeks. The 12% clonal frequency cutoff at Week 2 separated the clones that trended towards dominance (clones 01–3) versus withdrawal (clones 04–15). Error bars denote standard deviations of the six samples analyzed by flow cytometry for the day except for Days 51 and 72, on which two samples were analyzed. 1.35–2.32E6 cells were clonally assigned, per sample, on all days. (c) Mono-clonal growth curves of five of ct-MelaChroma’s participant clones. WT denotes un-transduced A375 cells. Four measurements were made per time point between two consecutive sub-cultures. Error bars indicate standard errors for each measurement.

Mentions: Clonal frequencies showed small standard deviations between the six ct-MelaChroma samples, particularly at early time points. Divergence of ct-MelaChroma’s participant clonal frequencies from their initial, matching values (100/15 = 6.67%) occurred early and quickly (Fig. 4a,b). Three dominant clones emerged (01, 02, 03), roughly doubling in clonal frequency in the first two weeks, while other clones (04–15) trended towards withdrawal. Notably, the most dominant clone (01) in the first half of the study exhibited frequency decline beyond Week 9. To determine if the observed pattern of clonal evolution is simply a reflection of individual clonal growth rate, we measured the growth of five participant clones in mono-clonal cultures (Fig. 4c). Interestingly, the fastest growing clone (04) was a withdrawing clone in ct-MelaChroma and its growth rate surpassed that of ct-MelaChroma’s dominant clone 01.


Defining Clonal Color in Fluorescent Multi-Clonal Tracking.

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

ct-MelaChroma’s clonal composition over time.(a) Mean frequency of participant clones of ct-MelaChroma on various days post pooling. Emergence dominance of three clones (01, 02, 03) was evident by Day 8. (b) Clonal composition of ct-MelaChroma over the course of fifteen weeks. The 12% clonal frequency cutoff at Week 2 separated the clones that trended towards dominance (clones 01–3) versus withdrawal (clones 04–15). Error bars denote standard deviations of the six samples analyzed by flow cytometry for the day except for Days 51 and 72, on which two samples were analyzed. 1.35–2.32E6 cells were clonally assigned, per sample, on all days. (c) Mono-clonal growth curves of five of ct-MelaChroma’s participant clones. WT denotes un-transduced A375 cells. Four measurements were made per time point between two consecutive sub-cultures. Error bars indicate standard errors for each measurement.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: ct-MelaChroma’s clonal composition over time.(a) Mean frequency of participant clones of ct-MelaChroma on various days post pooling. Emergence dominance of three clones (01, 02, 03) was evident by Day 8. (b) Clonal composition of ct-MelaChroma over the course of fifteen weeks. The 12% clonal frequency cutoff at Week 2 separated the clones that trended towards dominance (clones 01–3) versus withdrawal (clones 04–15). Error bars denote standard deviations of the six samples analyzed by flow cytometry for the day except for Days 51 and 72, on which two samples were analyzed. 1.35–2.32E6 cells were clonally assigned, per sample, on all days. (c) Mono-clonal growth curves of five of ct-MelaChroma’s participant clones. WT denotes un-transduced A375 cells. Four measurements were made per time point between two consecutive sub-cultures. Error bars indicate standard errors for each measurement.
Mentions: Clonal frequencies showed small standard deviations between the six ct-MelaChroma samples, particularly at early time points. Divergence of ct-MelaChroma’s participant clonal frequencies from their initial, matching values (100/15 = 6.67%) occurred early and quickly (Fig. 4a,b). Three dominant clones emerged (01, 02, 03), roughly doubling in clonal frequency in the first two weeks, while other clones (04–15) trended towards withdrawal. Notably, the most dominant clone (01) in the first half of the study exhibited frequency decline beyond Week 9. To determine if the observed pattern of clonal evolution is simply a reflection of individual clonal growth rate, we measured the growth of five participant clones in mono-clonal cultures (Fig. 4c). Interestingly, the fastest growing clone (04) was a withdrawing clone in ct-MelaChroma and its growth rate surpassed that of ct-MelaChroma’s dominant clone 01.

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