Input cell dose affects clone formation in xenotransplants

As a first experimental model of human breast cancer growth in vivo, we injected separate groups of highly immunodeficient female NOD/SCID/IL2rγ−/− (NSG) or NOD/RAG1−/−IL2rγ−/− (NRG) mice subcutaneously (on the back) with cells from two widely used human breast cancer cell lines: MDA-MB-231 and SUM-149 cells. To measure the number and size of clones present in the tumours subsequently generated, we exposed the cells just before the transplant with a previously described lentiviral library containing thousands of different DNA barcodes under conditions designed to obtain a single unique barcode in each cell transduced19. A total of 13 mice were injected subcutaneously with 2 × 104 to 50 × 104 cells, of which 30–35% were shown to be transduced (GFP+) and hence could be assumed to be individually barcoded11,19. Palpable tumours were apparent in all mice within 7 to 13 weeks, depending on the number of cells transplanted (Fig. 1). Approximately 10% of each harvested tumour was then used to determine the number and frequency of barcodes present using massively parallel sequencing of barcode-containing amplicons and a series of spiked-in controls included in each test sample (see Methods for details). The controls allowed us to set a conservative threshold for clone detection at a fractional read value (FRV) equivalent to 300 cells, based on the finding that this FRV allowed clones containing more than 500 cells to be detected at 90% sensitivity (9 of 10 control samples detected, with an inferred detection efficiency of 100% for clones of more than 5,000 cells) and an associated specificity of 99.7% (only one false-positive clone among 390 examined in 10 control cell samples, Supplementary Fig. 1 and Supplementary Table 1).

Figure 1: Experimental design. Serial passages of individual tumours derived from separate samples of barcoded MDA-MB-231 and SUM-149 cells (nine and four, respectively) are shown in purple, and for cells from patient-derived xenografts in green. White boxes indicate early passages of patient-derived xenografts, before barcoding cells for subsequent clonal analyses. The total number of cells (or fraction of the previous tumour) used to initiate each subsequent tumour, the time before removing the tumour for analysis, and the number and frequency of uniquely barcoded clones (expressed as a proportion of the estimated input number of barcoded cells) are shown on the right. CIC frequencies were calculated as the no. of clones divided by the total no. of barcoded cells transplanted based on the 30% transduction efficiency measured by FACS analysis of input cells. Full size image

Using this FRV threshold to define the presence of a clone, we then quantified the number of clones present in the tumours obtained (Fig. 1). In mice transplanted with MDA-MB-231 cells (M1–M9), the total number of barcoded clones thus detected in each tumour varied from 9 to 1,308. From these numbers and the number of transduced MDA-MB-231 cells originally transplanted into each mouse, we calculated the frequency of barcoded cells that had generated a detectable clone. Surprisingly, the ‘clone-initiating cell’ (CIC) frequency values thus obtained were not constant, but decreased over more than two orders of magnitude (from 1 in 7 to 1 in 3,000 of the cells transplanted) as an inverse function of the input cell dose over the 25-fold range of cell doses tested (from 2 × 104 to 50 × 104 total cells per mouse, Fig. 2a). Similar results were obtained for the tumours generated from 10 × 104 to 50 × 104 SUM-149 cells (S1–S4). In this case, the corresponding number of detectable clones decreased 15-fold (from 185 to 12), resulting in detectable CIC numbers that decreased from 1 in 160 to 1 in 12,500 of the cells transplanted.

Figure 2: Variation in clone size in primary and metastatic tumour xenografts. (a) An inverse linear relationship is seen between the numbers of cells transplanted and the number of clones detected, for both cell lines tested (Δ SUM-149 cells; ○ MDA-MB-231 cells). Values shown are the geometric mean±s.e.m. of the frequency of CICs calculated for each of the tumours identified in Fig. 1. (b) Cumulative distributions of clone sizes in primary xenografts generated from different numbers of S1–S4 (upper panel) and M1–M9 cells (lower panel). (c) Overlap between clones present in the tumour (M8) that arose at the site of injection (black) and in simultaneously assessed liver metastases (brown). (d) Comparison of the distributions of the 52 overlapping clones identified in panel c (upper panel) and for the 816 and 140 clones detected simultaneously at the injection site (black) and liver (brown), respectively (lower panel). The x-axis represents the size of clones binned in log 2 -increments. Full size image

Highly variant clonal growth of cell lines in vivo

The barcode data also allowed clone size measurements to be related to the source and dose of the cells transplanted. To accommodate the different sizes of tumours being compared, each clone size was normalized to the total size of the tumour in which it was present. Cumulative distribution plots of these normalized clone size values for each of the 13 tumours generated from the MDA-MB-231 and SUM-149 cells showed significant variation in the relative clone sizes produced both within and between tumours, but without a clear input cell dose effect (Fig. 2b). The largest range in clone size within a single tumour was observed in the M3 tumour (>104–fold). Between tumours, even the largest clones spanned a similar range despite similar inputs.

Macroscopically obvious metastases were noted in the liver of a single mouse that had been injected with the lowest dose (2 × 104) of MDA-MB-231 cells (tumour M8). Analysis of the number of unique barcodes detected in the bulk liver cells obtained from this mouse indicated the presence of 192 clones (Fig. 2c). Notably, the majority (140) of the 192 metastatic clones were not detected in the tumour that arose subcutaneously at the initial site of M8 cell injection, and were relatively small, ranging in size from 103 to 4 × 104 cells per clone (Fig. 2d). The other third of the metastatic clones arose from cells whose progeny were readily detected in the tumour that arose at the site of injection. They also reflected the same bimodal clone size distribution (with upper and lower modal clone size values at both sites differing by a factor of ~10), although, on average, those present in the liver were again smaller (Fig. 2d).

These results demonstrate the sensitivity of vector-mediated barcoding to detect and monitor a wide range of CIC activity that can be displayed by malignant human cells proliferating in tumour xenografts and derived metastases. They also show that, at least for the cell lines tested here, manifestation of CIC activity is highly negatively affected when the number of cells initially injected is in the range commonly used.

Passaged cell lines exhibit changing clonal growth patterns

To monitor the stability of the diverse clonal growth activity exhibited by the MDA-MB-231 and SUM-149 cells that generated primary tumours, we transplanted 10% of the cells harvested from four of the primary tumours (M3, M4, S3 and S4) into secondary mice. In each case, palpable tumours were detected 3 to 5 weeks later, at which time these secondary tumours were harvested for analysis of their barcode content, except for M4, where 10% of the cells were used to generate tertiary tumours (Fig. 1). The barcode data obtained from these passaged tumours indicated that they contained consistently fewer clones than the primary tumours from which they had been derived, although in the matched pairs (derived from the same parental tumour), the reduction in content of barcoded cells was similar (approximately twofold for the passaged cells in the M3 and M4 experiments, and approximately sixfold for the passaged cells in the S3 experiment). However, both the frequency of all clones that were detectable at every passage and their individual prominence was highly variable.

To enable a more comprehensive comparison of the growth patterns exhibited by different clones in sequentially generated tumours, we used k-means clustering to group clones according to the measured change in size of each on sequential passaging (Supplementary Fig. 2). For the M3 experiment, this analysis identified three general patterns (Fig. 3a). The most prevalent of these (displayed by 62% of all M3 clones detected) was one where the relative clone size remained constant. The least prevalent pattern (displayed by 18% of all M3 clones detected) was one where the relative clone size decreased in the second passage. The remaining 20% of all M3 clones detected displayed a pattern where the relative clone size increased with passaging (Fig. 3b). Within each of these groups, additional subgroups were also resolved according to the relative size of each clone and the magnitude of its change in size (Fig. 3a).

Figure 3: Diverse in vivo clonal growth patterns of human breast cancer cell lines. (a) Growth patterns of individual clones in primary and secondary derivative tumours generated from M3 cells. In each plot, a separate line portrays the growth activity of an individual clone in successive passages. Clones that remained relatively constant in size between passages are shown in shades of red, and those whose size increased or decreased are shown in shades of yellow and blue, respectively. The area in each plot shaded in grey represents the relative clone size below the threshold used for detecting barcoded clones. In cases where replicate tumours had different limits of detection, and are represented on the same plot, the higher limit is shown. (b) Relative proportions of the different clonal growth patterns at each passage from M3, S3 and S4 cells. Colours in each sector correspond to the colour-coded clonal patterns described in a. Full size image

For the M4 experiment, the k-means cluster analysis identified the same three groups but with fewer subgroups (Fig. 4a) and the prevalence of their different growth patterns was reversed. Thus, the most prevalent pattern (displayed by 57% of all M4 clones detected) was for the clones that became smaller after the first transplant, and the least prevalent pattern was where clones remained constant or increased in size (6 and 4%, respectively, of all M4 clones detected, Fig. 4). Interestingly, in this experiment, k-means clustering identified two additional groups, both characterized by delayed clonal growth; that is, clones that were not detectable until the formation of secondary, or even tertiary tumours, and these comprised 10 and 23%, respectively, of all clones identified. Notably, many of the clones that became detectable in the secondary tumours, subsequently, decreased in tertiary tumours, indicative of a fluctuating growth pattern.

Figure 4: Delayed growth of M4 clones in serially transplanted mice. (a) Growth patterns of individual clones in primary, secondary and tertiary tumours derived from M4 cells. In each plot, a separate line portrays the growth activity of an individual clone in successive passages. Clones that remained relatively constant in size between passages are shown in shades of red, and those whose size increased or decreased are shown in shades of yellow and blue, respectively. Clones that first became detectable in secondary and tertiary tumours are shown in grey and black, respectively. The area in each plot shaded in grey represents the relative clone size below the threshold used for detecting barcoded clones. In cases where replicate tumours had different limits of detection, and are represented on the same plot, the higher limit is shown. (b) Relative proportions of the different M4 clonal growth patterns at each passage. Colours in each sector correspond to the same colour-coded clonal patterns described in a. Full size image

In the S3 and S4 experiments, seven and six clusters, respectively, were identified, and these also included clones whose sizes remained constant, expanded or diminished (Fig. 3b and Supplementary Fig. 3) and their proportions quite similar in both experiments: 33, 50 and 17% of all clones detected, respectively, in experiment S3, and 38, 30 and 32% of all clones detected in experiment S4.

These findings illustrate the unbiased and strong resolving power of DNA barcoding to reveal distinct patterns of clonal growth in a complex landscape of behaviour obtained in highly polyclonal xenografts of malignant human cell lines.

Diverse in vivo dynamics of CICs from patients’ tumours

We then conducted a similar series of experiments with cells obtained from xenografts of three patients’ breast cancers (Fig. 1). One of these xenografts had been generated from a pleural effusion that developed in a patient with an advanced stage (originally ERα+) tumour. In this case, the cells to be barcoded were obtained from a primary xenograft (T1). Barcoding was also applied (separately) to cells from a secondary and a tertiary xenograft originally generated from another primary ER−PR−HER2+ tumour (T2), as well as to a tertiary xenograft of cells originally from a third, primary ER−PR−HER2+ tumour (T3)10. Aliquots of 0.7 × 105 to 10 × 105 cells from these xenografts were injected into NSG mice immediately post-transduction and the efficiency of transduction shown to be ~30%, based on the proportion of cells that were GFP+ in an aliquot kept in culture for another 2 days. Tumours derived from the barcoded cells became evident 9 to 16 weeks post-transplant. Each of these was then passaged another 2–3 times with paired replicates, as indicated in Fig. 1.

In the 10% sample removed from the cells harvested from the first of the tumours thus generated, we identified 6 × 106 to 9.6 × 108 barcoded cells and these were distributed among 125 to 2,190 different clones. CIC frequencies calculated from these clone numbers (divided by the number of barcoded cells injected) varied over a >100-fold range (from 1/11 to 1/2,400 cells transplanted) for the different tumour sources, although values were quite consistent between paired replicates. Interestingly, the total number of clones detected in each tumour decreased in later tumour passages, as had been observed for the tumours generated from the human breast cancer cell lines.

We also used k-means clustering to determine the number and types of different clonal growth patterns obtained in these patient tumour-derived xenografts (Supplementary Fig. 4). Analysis of the data from the T1-11 and T1-12 tumours showed these shared only three of the patterns exhibited by the serially passaged cell lines; that is, no change, a decreasing, or a fluctuating change in clone size, and did not include clones that increased in size or showed a delayed onset of growth (Fig. 5a and Supplementary Fig. 5). There was also a marked variation in the relative prevalence of each pattern in T1-derived clones. For example, clones that fluctuated in size were highly prevalent in T1-11 (61% of all clones in that series), whereas, in T1-12, clones that diminished in size were the most prevalent (69%, Fig. 5b). Moreover, approximately one-third of the T1-11 clones that displayed a fluctuating behaviour were present at a detectable level in the first tumours studied, but then diminished in the next passage and reappeared in a subsequent passage. The other fluctuating T1-11 clones exhibited an opposite pattern in which they were not detected initially but became detectable in the next passage and then subsequently decreased, or first became detectable in the third passage. In contrast, for T2, a continuous increase or decrease in clone size was the most prevalent patterns seen (displayed by 45 and 54%, respectively, of T2-111 clones, and 53 and 41%, respectively, of T2-1121 clones, Fig. 5b and Supplementary Fig. 6). Thus, the presence of fluctuating clones and the high variability in prevalence of different growth patterns between replicates for T1 more closely resembles the behaviour of MDA-MB-231 cells, whereas the presence of only three main growth patterns and their relative reproducibility between replicates for T2 resembles the behaviour of SUM-149 cells.

Figure 5: Diverse in vivo clonal growth patterns of patient-derived tumour xenografts. (a) Growth patterns of individual clones in primary, secondary and tertiary tumours generated from T1-11 cells. In each plot, a separate line portrays the growth activity of an individual clone in successive passages. Clones that remained relatively constant between passages are shown in shades of red, and those whose size decreased are shown in blue. Clones that first became detectable in secondary tumours or that fluctuated in size between passages are shown as grey. The area in each plot shaded in grey represents the relative clone size below the threshold used for detecting barcoded clones. In cases where replicate tumours had different limits of detection, and are represented on the same plot, the higher limit is shown. (b) Relative proportions of the different clonal growth patterns exhibited by T1-11 and T1-12 cells. Colours in each sector correspond to the colour-coded clonal patterns described in a. (c) Relative proportions of the different clonal growth patterns exhibited by T2-111 and T2-1121 cells. Colours in each sector correspond to the colour-coded clonal patterns described in a, except for clones that increased in size between passages that are shown in yellow. Full size image

These results reveal the extensive heterogeneity in the clonal growth dynamics seen both within and between tumour xenografts derived from different minimally xenografted human breast cancer cells from patients, as well as some similarities to the two human breast tumour cell lines studied.

Asymmetry of growth activity in paired replicate tumours

To evaluate the extent to which these varying clonal growth dynamics might reflect random events, we compared the patterns obtained in pairs of secondary tumours generated from the same primary tumour (that is, from M3, M4, S3, T1-11, T1-12 and T2-1121). These paired comparisons showed that the majority of replicate clones (60–98%) were symmetrical in their subsequent growth patterns (Fig. 6). Experiments M3 and M4 demonstrated the most asymmetry in growth activity (33 to 40% of replicate clones showed different growth trajectories, Fig. 6a), and this usually occurred when the progeny of a clone increased or decreased in size in one daughter tumour only. Interestingly, the clones that most frequently displayed a symmetrical behavior in paired derivative tumours were those whose initial growth was delayed (~90% of the clones that were first detected in secondary tumours showed a replicate clone first detected in tertiary tumours).

Figure 6: Replicate xenografts include both symmetric and asymmetric clonal growth patterns. (a) Venn diagrams showing the proportion of clones that demonstrated symmetrical (non-overlapping parts of the circle), and asymmetrical growth patterns (overlap between two circles) in replicate tumours derived from parental M3, M4 and S3 tumours. Different colours are used to identify each of the five clonal growth patterns detected as follows: constant (red), increasing (yellow), diminishing (blue), fluctuating (first appearing in secondary tumours, grey) and delayed (first appearing in tertiary tumours, black), and the size of each circle reflects the relative abundance of clones displaying the growth pattern it represents. The numbers shown are the absolute numbers of clones whose replicate derivatives displayed symmetrical or asymmetrical growth patterns. (b) Venn diagrams showing the proportion of clones that demonstrated symmetrical (non-overlapping parts of the circle) and asymmetrical growth patterns (overlap between 2 circles) in replicate tumours derived from parental T1 and T2 tumours, using the same colour coding and illustrative principles as in panel a. Full size image

In the patient-derived xenografts from tumour T1, 80 to 98% of replicate clones showed symmetry of growth activity, and the majority of asymmetry was displayed by clones whose initial size subsequently decreased in one derivative tumour and fluctuated in the other (44 to 60% of all fluctuating clones, showed a decline in the replicate tumour, Fig. 6b). In the xenografts derived from T2, 98% of the clones that maintained a relatively constant size between passages in one replicate showed a declining size in the other.

These analyses demonstrate a strong similarity in the growth behaviour of many clones as assessed in their paired derivatives, although examples of all possible combinations were also detected (Fig. 6).