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Increased bone metastases rates and sizes can be achieved by introducing these cell lines via intra-cardiac
injection. Osteolytic bone metastases can be detected in 70%-90% of mice following injection of 4T1-2,
E0771 or Py8119 cells into the left cardiac ventricle, however lung and other soft tissue metastases are still
prevalent in all of these model systems [60-62] . It therefore appears that the only way to model bone metastases
in syngeneic model systems, without the complications associated with soft tissue metastases, is to inject
tumour cells directly into the long bones (tibia/femur). Injecting mouse mammary cancer cells into their
complementary mouse model results in high engraftment rates (up to 100%) and provides a useful tool for
modelling tumour cell-bone cell interactions in an immune competent environment [60,61,63] .

ASSESSMENT OF TUMOUR PROGRESSION IN BONE
Imaging in live animals
To enable analysis of tumour progression at the primary site and/or in bone in live animals, cell lines/PDXs
are commonly pre-labelled with luciferase or fluorescent proteins before injection. Fluorescently-labelled
tumours [Figures 2 and 3A] can be detected by placing the mouse under a light source and visualising
[36]
through the correct filters using a simple machine such as a LightTools box. This method is cheap, quick
and easy to use; however, the data are not quantifiable, and skeletal tumours can be difficult to observe until
they are very large due to autofluorescence from the bone and light scatter. Bioluminescent imaging is much
more sensitive, and tumours in bone can be detected using this method before they are visible on
histological sections. This method therefore allows longitudinal, real time imaging studies of metastases
development in bone. Importantly, data obtained from bioluminescence readouts [6,42,43] from tumours using
a luminomior such an IVIS system are quantifiable, making this the preferred analytical method.

In addition to tumour growth, researchers are often interested in the effects that tumours and/or treatments
are having on the bone microenvironment. Osteolytic lesion area and abnormal bone remodelling can be
visualised and assessed weekly in vivo using a cabinet X-ray machine or by live µCT imaging of the affected
bone(s) [Figure 3B] [18,42,43,64] . Using in vivo µCT scanning, percentage changes in trabecular bone parameters
and the size or number of osteolytic lesions can be measured over time as bone disease
develops [Figure 4A-C]. In addition, following bone modulating therapy, effects on bone repair (new bone
formation and bone density) can be mapped by overlaying bone images from the same mouse at different
time points, as previously described [65,66] .

Post-mortem analysis of bone metastases
With the exception of metastases from PDXs, bone lesions develop rapidly in mice, and animals should be
monitored daily for changes in activity levels, mobility and onset of cachexia. For humane endpoint, mice
should be euthanised before 20% body weight is lost, tumour progression impairs mobility or an animal
appears to be in respiratory distress, taking into consideration local guidelines/approvals. To ensure that
data obtained from experiments are comparable between groups, mice should be culled on the same day so
that the experimental timeframe is standardised, the exception being for experiments designed to assess
survival.

On termination of the experiment, mice should be examined closely for evidence of metastatic foci outside
of bone. For studies in which tumour cells have been introduced via intra-cardiac injection, researchers
should check accuracy of their technique before determining which mice should be selected for downstream
analysis. For all tumour cell types, growth in the mediastinum surrounding the heart indicates that cells
were not accurately injected into the left cardiac ventricle, and, specifically for MDA-MB-231 cells, tumour
growth in the lungs suggests mis-injection into the right cardiac ventricle, from which bone metastases will
not develop. Mice in which these mis-injections are detected should be excluded from the study. On