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

[15]
with bone homing . Injection into the lateral tail vein may better model bone homing, as tumour cells
introduced into the venous circulation in this manner need to make at least one pass through the circulatory
system before being disseminated in bone. Indeed, introduction of most cell lines into the lateral tail vein
results in tumour growth in the lungs and only cells “homed” to bone produce bone metastases with this
method . However, studies comparing specific molecular profiles of MDA-MB-231 cells that for form
[37]
bone metastases following intra-cardiac injection (MDA-MB-231-B02) and clones of MDA-MB-231 cells
that form bone metastasis following intra-venous injection (MDA-MB-231-IV) have demonstrated that
both of these clones show bone homing profiles and that these clones are more closely related to each other
than to the parental clone [15,19,30] . Furthermore, intra-cardiac and intra-venous injection of bone homing
clones of MDA-MB-231 cells results in tumour cell dissemination into the same metastatic niches
(endosteal and peri-vascular) indicating that both models are equally as useful for studying processes
associated with early dissemination into bone but may be less useful for identifying mechanisms associated
[38]
with bone homing .

In breast cancer patients, it is believed that tumour cells at the primary site “prime” the bone for arrival of
disseminating tumour cells, facilitating metastasis to this site . This process is difficult to model in mice as
[39]
human breast cancer cell lines rarely, if ever, spontaneously metastasise from the mammary fat pad to the
mouse skeleton. Researchers have recently developed ER-positive and ER-negative models of spontaneous
breast cancer metastasis to bone. In these models, injection of MCF7 or T47D cells into the fourth
mammary fat pads results in spontaneous metastasis to mouse bone in ~50% of animals [6,23,40] . In addition,
injection of ER-positive BB3RC2 and BB2RC08 PDXs or ER-negative BB6RC37 PDXs into the fourth
mammary duct using the MIND model also results in spontaneous metastasis to mouse bone in 32%, 20%
[23]
and 20%. respectively . Interestingly, in all of these models, mice were supplemented with oestradiol to
support tumour growth. In the MCF7 and T47D models, development of overt bone metastases was
dependent on mice being supplemented with oestradiol, irrespective of the growth of the primary tumour;
whether the same is true for these particular PDX models remains to be determined. Because oestradiol has
profound bone anabolic effects, especially in mice [Figure 1], and can also alter anti-cancer immunity,
supplementation with this hormone adds complexity to analysing results obtained using these models [2,40] .

(3) Breast cancer dormancy models

As metastasis to mouse bone primarily occurs in young, 4 to 8-week-old animals or following oestradiol
supplementation, it is hypothesised that high bone turnover is necessary to stimulate growth of human
cancer cells in mouse bone. Researchers have used this idea to generate models in which manipulation of
the bone environment can be used to investigate factors associated with breast cancer cell dormancy and
metastatic outgrowth. Dissemination of MDA-MB-231 breast cancer cells into bone via intra-cardiac
injection results in tumour cells lodging in the bone metastatic niche (primarily close to the growth plate in
the trabecular region on bone) [38,41] . In adult mice, metastatic outgrowth is rare, possibly due to low rates of
bone turnover, leading researchers to hypothesise that older “adult” mice may provide a useful model for
studying dormancy . Labelling tumour cells with lipophilic membrane dyes, such as 1,1-Dioctadecyl-
[42]
3,3,3,3-tetramethylindodicarbocyanine (DiD) and 1,1’-Dioctadecyl-3,3,3’,3’-Tetramethylindocarbocyanine
Perchlorate (DiL), before intra-cardiac injection enables the researcher to differentiate between dormant
and proliferating cancer cells in bone as the dyes are retained in the membrane of non-proliferating
(dormant) cells but are progressively lost as cells go through subsequent rounds of proliferation [38,42,43] . Using
DiD labelled MDA-MB-231 cells, researchers have demonstrated that in adult (14-16 weeks old) mice
tumours lodge in the bone but remain dormant in this site with metastatic outgrowth occurring in > 10% of
animals over the 84-day time period tested . Stimulating osteoclastic bone resorption by ovariectomy
[43]