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Page 6 of 20 Ottewell et al. J Cancer Metastasis Treat 2021;7:11 https://dx.doi.org/10.20517/2394-4722.2021.14

Considerable amounts of research are therefore being carried out to produce alternative models that can be
used to answer questions that are less suited to the traditional, MDA-MB-231, intracardiac model. Indeed,
ER-positive MCF7 and T47D models that display long latency may be more representative of human
disease and may prove to be useful for investigating efficacy of treatments on mixed lesions, as this
phenotype is not commonly investigated in mouse models but is observed in a subset breast cancer
[27]
patients . With careful selection of the appropriate model systems researchers now have a fairly
comprehensive tool set for investigating various aspects of breast cancer bone metastasis [Table 1].

(1) Improving outcome, frequency and molecular subtypes represented in mouse models of breast cancer
bone metastases

To increase the frequency of bone metastases and reduce the need for intra-cardiac injections, bone seeking
sub-lines of MDA-MB-231 cells have been produced via repeated in vivo passaging through mouse
bones [7,15,19,28] ; MDA-MB-231-B02, MDA-MB-231-B and MDA-MB-231-bone cells form tumours in up to
90% of mice following injection into the caudal artery, whereas MDA-IV cells form tumours in 80%-90% of
mice after injection into the lateral tail vein [7,15,19,28-31] . Via a similar mechanism, of in vivo passaging,
researchers have also produced a bone seeking MCF7 breast cancer cell line that predominantly forms
osteolytic lesions after 10-12 weeks . Recently, researchers have reported high incidences of bone
[26]
metastasis from ER-positive MCF7 cells following injection via the caudal artery without the need for
oestradiol supplementation, suggesting that this method may be useful for modelling bone metastases from
a wide variety of breast cancer subtypes . Injecting tumour cells via the caudal artery/tail vein appears to
[25]
result in bone metastases primarily forming in the hind limbs, simplifying downstream analysis and
allowing reduced numbers of animals to be used for statistical analysis. Importantly, these methods are not
associated with stroke or early hind limb paralysis, reducing risks of adverse events that are commonly
observed following intra-cardiac injection.

Many breast cancer cell lines do not form bone metastases following injection into the blood stream. For the
majority of these, bone metastases can be modelled by direct injection into the tibia or femur, which results
in the development of osteolytic tumours in the corresponding bone. This method has successfully been
used to model interactions between the bone microenvironment and tumour cells and has shown efficacy as
a method for xenografting ER-negative MDA-MB-231, MDA-MB-436 and SUM 1315 cells as well as PDXs
and ER-positive MCF7 or T47D cells following oestradiol supplementation [32-36] . This method has the
advantage of high tumour take rates in bone and is useful for studying genetic manipulation of the
host/tumour cell environment. However, intra-osseous injection results in considerable damage to the bone
cortex and displaces cells within the marrow cavity promoting increased cytokine release and bone turnover
during the healing process; these factors must be taken into account when analysing data acquired using this
method. Furthermore, intra-osseous injection bypasses the early stages of metastasis including homing to
the bone microenvironment and tumour cells do not need to interact with the bone metastatic niche from
which bone metastases normally develop. Intra-osseous injection cannot therefore be used to model tumour
cell dormancy or early stages of metastases.

(2) Modelling bone homing with human breast cancer xenografts

Injection of breast cancer cells into the left cardiac ventricle or caudal artery results in tumour cells being
disseminated into the skeleton via the arterial system. It has been argued that these cells do not home to
bone; instead, tumour cells become trapped in the capillaries, from which they then migrate to the
metastatic niche(s). Therefore, this is not an appropriate model for investigating mechanisms associated