Evidence for clonal dominance: comparison of the distributions of HCT-116 barcoded clones and cells by clone size category <i>in vitro</i> and <i>in vivo</i><b>.</b>

<p>For each graph, the number of independent clones (blue bars) or the total aggregate number of cells (orange bars) is plotted for each clone size category across the X axis. (A) Distribution of HCT-116 barcodes <i>in vitro</i>: 80% of the barcodes (clones) were identified in categories “128–255” to “2048–4095”, indicating that 80% of the cells divided between 7 and 12 times; 95% of the cells were identified in categories “128–255” to “2048–4095”, indicating that 95% of the cells belong to clones derived from 80% of the originally transduced cells, which divided between 7 and 12 times. Scale for number of cells was adjusted 500 fold to allow side by side comparisons of clone numbers and cell numbers. (B) Distribution of HCT-116 barcodes <i>in </i><i>vivo</i>: 75% of the barcodes are found in categories “missing” to “2–3”, indicating that they either didn’t survive at all, didn’t divide, or divided no more than once and represented only 1% of the retrieved tagged cells. However, only 6% of the bar-codes were located in categories “64–127” to “16384–32767”, indicating that they divided between 6 and 14 times; 95% of the cells accrued in categories “64–127” to “16384–32767”, indicating that 95% of the tagged cells are derived from 6% of the initially barcoded clones that divided the most. Scale for number of cells was adjusted 25 fold to allow side by side comparisons of clone numbers and cell numbers.</p

Barcode recovery rate from HCT-116 cells in NSG mice and <i>in vitro</i>.

<p>Barcode recovery rate from HCT-116 cells in NSG mice and <i>in vitro</i>.</p

Evidence that encoded shRNA did not influence HCT-116 clonal cell growth <i>in vivo</i>.

<p>(A) Distribution of the 4,109 unique barcodes retrieved most abundantly from the three xenograft tumors (marked with red vertical lines) across the entire shRNA population distribution in the shRNA library. (B) Detection of a large fraction of the most abundant barcodes from the three xenograft tumors (marked in red) in the shRNA population most toxic to HCT-116 upon Doxycycline induction <i>in vitro</i> for 15 days (measured by a negative Log2 ratio of mean read counts for individual shRNA at Day 15 following Doxycycline to mean read counts at Day 0).</p

Barcode recovery rate in Athymic Nude xenograft experiments.

<p>Barcode recovery rate in Athymic Nude xenograft experiments.</p

Evidence that encoded shRNA are not induced in the absence of Doxycycline <i>in vitro.</i>

<p>(A) A lentiviral library containing 27,500 unique barcodes was used to transduce HCT-116 cells at a MOI of 0.3. After 72 hours, of puromycin selection, cells were continuously passaged for 15 days in the absence of Doxycycline. Day 0 and Day 15 cell aliquots were obtained and total DNA from both time points were subjected to the barcode high-throughput sequencing retrieval procedure. (B) Correlation plot for Day 15 measurement against Day 0. shRNA barcode reads for all samples from all time points were first normalized to 2×10⁷ reads. Values for triplicate samples were averaged. At Day 15, strong repression of shRNA expression in the absence of Doxycycline is evident in the correlations with the Day 0 reference. Plot of log10 mean normalized reads for Day 0 against Day 15 (Pearson correlation: R = 0.99).</p

Percentage of transduced cells at a given MOI.

<p>Percentage of transduced cells at a given MOI.</p

Measurement of Cancer Cell Growth Heterogeneity through Lentiviral Barcoding Identifies Clonal Dominance as a Characteristic of <i>In Vivo</i> Tumor Engraftment

<div><p>Advances in the fields of cancer initiating cells and high-throughput <i>in vivo</i> shRNA screens have highlighted a need to observe the growth of tumor cells in cancer models at the clonal level. While <i>in vivo</i> cancer cell growth heterogeneity in xenografts has been described, it has yet to be measured. Here, we tested an approach to quantify the clonal growth heterogeneity of cancer cells in subcutaneous xenograft mouse models. Using a high-throughput sequencing method, we followed the fate <i>in vitro</i> and <i>in viv</i>o of ten thousand HCT-116 cells individually tagged with a unique barcode delivered by lentiviral transduction. While growth <i>in vitro</i> was less homogeneous than anticipated, we still find that 95% of the final cells derived from 80% of the original cells. In xenografts, however, 95% of the retrieved barcoded cells originated from only 6% of the initially injected cells, an effect we term “clonal dominance”. We observed this clonal dominance in two additional xenograft models (MDA-MB-468 and A2780^cis) and in two different host strains (NSG and Nude). By precisely and reproducibly quantifying clonal cancer cell growth <i>in vivo</i>, we find that a small subset of clones accounts for the vast majority of the descendant cells, even with HCT-116, a cell line reported to lack a tumor-initiating compartment. The stochastic <i>in vivo</i> selection process we describe has important implications for the fields of <i>in vivo</i> shRNA screening and tumor initiating cells.</p></div

Tracing individually-labeled HCT-116 cancer cell clones <i>in vitro</i> and <i>in vivo.</i>

<p>A lentiviral library containing 27,500 unique barcodes was used to transduce HCT-116 cells at an MOI of 0.1. Pools of 10⁵ cells, corresponding to 10⁴ individually tagged cells were either admixed with 3×10⁶ cells and implanted subcutaneously into NSG mice or grown in culture <i>in vitro</i>. Cells were harvested after 9 days and tumor removed after 12 days, and total DNA preparations from cells or tumors were subjected to the barcode retrieval procedure.</p

Percentage of cells carrying n lentiviral insertions for a given MOI.

<p>Percentage of cells carrying n lentiviral insertions for a given MOI.</p

DataSheet1_The 10th Santorini conference: Systems medicine, personalised health and therapy. “The odyssey from hope to practice: Patient first. Keep Ithaca always in your mind”, Santorini, Greece, 23–26 May 2022.DOCX

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