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Analysis of surface tumour burden is useful in providing a whole (albeit superficial) view of metastatic colonization on a per-lymph node basis over progressive time points. To study the growth of metastatic deposits that extend beyond the subcapsular sinus, however, analysis of the histological cross- sections of the lymph node was performed. Figure 2.4A-D show representative histological cross-sections taken at each time point (low magnification, 25X). Although the presence of tumour cells was confirmed at higher magnification (1000X) at 90 minutes and 3 days post-injection (see insets of Figure 2.4A-B), histological tumour burden at low magnification did not become apparent until 7 and 14 days, as seen in panels C and D, respectively. This is due to the diminished sensitivity of the point count method in detecting single tumour cells at low magnification. Only when tumour deposits grow larger than 0.01 mm2 does the probability of a grid point falling on tumour tissue (and being enumerated) increase. The changes in histological tumour burden over time are shown in Figure 2.4E. The means and standard deviations are shown, where n = 10 per group. The means of transformed data was analyzed by one-way ANOVA (p <0.001), and a post-hoc Tukey’s multiple comparisons test was used to determine which groups were significantly different from each other (* p < 0.5, *** p < 0.001).

Figure 2.4 Quantification of time-dependent changes in histological tumour burden. Panels A-D show histological sections at low magnification (25X) that are representative of their respective time points. Scalebar = 1 mm. In A-B, although tumour burden was not apparent at low magnification, tumour cells are seen to be arrested in the subcapsular sinus at high magnification (1000X, see insets). Arrow heads denote the location of tumour cells. Scalebar = 20μm. Although single tumour cells are difficult to detect at 25X magnification, tumour burden can be quantified by the point count method at 25X magnification when micrometastases (an example shown in panel C) and overt metastases (an example in panel D) form. Histological tumour burden between the time groups are quantified in E, where untransformed mean and back transformed standard deviations are shown, n = 10 per group. The means of transformed data (asin square root) was analyzed by one-way ANOVA (p < 0.001), and a post-hoc Tukey’s multiple comparisons test was used to determine which groups were significantly different from each other (* p < 0.05, *** p < 0.001).

2.3.5 Consistent delivery of reference beads in all time-end point groups

The role of 16 μm reference beads is central to LEMA for two important points: (1) the observations on lymph node tumour burden over progressive time intervals are presumably due to an actual biological phenomenon rather than technical issues such as an inconsistent delivery of injectate between groups of mice, (2) the accuracy of quantifying tumour cell fate by the “cell accounting” technique hinges upon the reference beads arresting in the same anatomical regions as tumour cells due to their similarity in sizes. Both of these assumptions imply that the average number of reference beads do not significantly vary between groups of mice. Therefore to determine if these assumptions are correct, the average number of reference beads per section was counted in six histological sections spaced 25 μm apart from each other. The graph in Figure 2.5 C demonstrates that the average number of reference beads per section did not significantly vary from 90 minutes, 3, 7 and 14 days. Furthermore, from these values we extrapolated the number of reference beads per entire volume of lymph node and found the values did not significantly vary from each other at any of the time points. Therefore we can conclude the two assumptions in LEMA are correct. One should note, however, there were 2 cases were where reference beads were absent in histology sections. In one case, a mouse in the 3 day time point, where the lymph node harbored only a few scattered isolated tumour cells. The other case was in day 7 in a mouse where the lymph node was negative for

metastasis. Slides containing lymph node sections that did not contain reference beads could be due to sampling error when sectioning the lymph node during histological preparation.

Figure 2.5 Quantification of the average number of reference beads delivered per lymph node for all time points. Panel A depicts an example of an area (subcapsular sinus, dashed box) where tumour cells and reference beads first arrest in the axillary lymph node. Scalebar = 2 mm. In B, a higher magnification of the dashed box area in A is shown. Arrow points to a reference bead, arrow heads denote tumour cells. Scalebar = 20μm. The mean number of reference beads per section was averaged from six sections per axillary lymph node per mouse. In panel C, the average of the means from all mice per time point are shown, error bars represent the standard error of the mean. The means were shown to not significantly vary between all time points, one-way ANOVA (p > 0.05). The numbers of mice enumerated per time point were 10, 9, 9 and 10, at 90 minutes, 3, 7 and 14 days, respectively.

2.3.6 Assessment of tumour cell survival and fate with respect to formation of micrometastases and overt metastases

In determining the fate of tumour cells after they arrest in the lymph node, we assumed that various types of metastatic deposits (isolated tumour cells, micrometastases, and overt metastases) are clonal in origin (Fidler and Talmadge, 1986). With this assumption, the percentage of tumour cells in the lymph node that have survived as a particular type of metastatic lesion was calculated. The percent tumour cells delivered to the lymph node that were present as isolated tumour cells, micrometastases, and overt metastases at the various time points is shown in Figure 2.6 A, B, and C, respectively. These survival data are summarized in the fate map in Figure 2.7, where stages of lymph node metastasis development are arranged in chronological order. 97% of the tumour cell injectate survived as isolated tumour cells at 90 minutes post- injection. Strikingly, tumour cell survival dropped from 97% to 7% from 90 minutes to 3 days. Single tumour cells were no longer found in the axillary lymph node beyond 3 days. Micrometastases that have formed at 3 days may contribute to the formation of overt metastases observed at 7 days post-injection. Also, some of the micrometastases at 3 days may have remained dormant, which may explain their presence at 7 and 14 days. In addition, there is a larger percentage of the originally delivered cells present as micrometastases at 3 days (0.36%), than detected as overt metastases at days 7 and 14, suggesting inefficiency in conversion from micrometastases to overt metastases.

Figure 2.6. Quantification of the fate of tumour cells in the lymph node. In panel A, the percentages of the tumour cells in the lymph node that survived

as isolated tumour cells for each time point are shown. Means and standard errors are shown. Means were compared by parametric one-way ANOVA in graph A (p < 0.0001), and Tukey’s multiple comparisons test was used to determine which means were significantly different (***,p < 0.001). Panel B shows the percentage of the tumour cells in the lymph node that formed micrometastases. In panel C, at 7 and 14 days post-injection, the proportion of tumour cells in the lymph node that were able to successfully form overt tumours was 0.002% and 0.08%, respectively. Means and standard errors are shown. In graphs B & C, means were compared by non-parametric ANOVA (Kruskal-Wallis test) where p < 0.001 and p < 0.01, respectively. Dunn’s multiple comparisons test was used to determine which means were significantly difference (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Figure 2.7. A flow chart showing the fate of tumour cells after they arrest in the axillary lymph node. Note the rapid loss of isolated tumour cells over time. The micrometastatic compartment also showed progressive loss from 3 days to 14 days post-injection. At 7 days, 0.002% of the tumour cells that arrested in the lymph node had formed overt metastases, and at 14 days, 0.08% of the tumour cells had formed overt metastases. Dotted arrows denote possible fates of the metastatic lesion. From this flow chart, it is apparent that there are several major sources of metastatic inefficiency in the lymph node: 1) there was a significant loss of isolated tumour cells and 2) only a small proportion of isolated tumour cells were able to form micrometastases, and 3) only a small proportion of micrometastases were able to successfully form overt metastases.

2.3.7 Sinus histiocytes as a possible source of tumour cell toxicity in the