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Page 10 of 19 Maurizi et al. J Cancer Metastasis Treat 2021;7:35 https://dx.doi.org/10.20517/2394-4722.2021.74

In bone metastases, osteoblasts and osteoclasts are reprogrammed by the tumor cells directly from the
primary site, through the release of EVs containing an osteotropic cargo. With regards to prostate cancer,
[127]
first studies from Itoh et al. showed that EVs derived from different prostate cancer cell lines stimulate
osteoblast differentiation of MC3T3 cells by releasing v-ets erythroblastosis virus E26 oncogene homolog 1,
which is an osteoblast differentiation-related transcriptional factor. Similarly, it has been demonstrated that
different prostate cancer cell line-derived exosomes shuttle phospholipase D2, which targets the osteoblasts,
stimulating their proliferation and differentiation by activating the ERK1/2 pathway . This effect is lost
[128]
[128]
when prostate cancer-derived exosomes are generated in the presence of a PLD pan inhibitor .
Interestingly, a bidirectional cross-talk between prostate cancer and osteoblast is conceivable, as
[129]
demonstrated some years ago by Millimaggi et al. , who found that osteoblast-derived conditioned media
stimulated membrane vesicle shedding in prostate cancer cells.

microRNAs are typical cargo of EVs and several reports showed their crucial role in PMN formation. Their
shuttling in the bone microenvironment by means of EVs can drive bone metastasis development towards
an osteolytic or an osteosclerotic phenotype. Indeed, Probert et al. recently demonstrated the delivery of
[130]
a set of RNAs by prostate cancer-derived EVs to osteoblasts, which positively regulates osteoblast behavior.
This mRNA cargo is enriched in genes related to cell surface signaling, cell-cell interaction, and protein
translation. Among the non-coding RNA, the authors also found an enrichment in miRNA-21, which has
already been linked to ovarian cancer exosome-mediated development and could be considered a
[131]
prognostic factor in the urine and plasma of prostate cancer patients [132,133] . Consistently, it has been
demonstrated that hsa-miR-940 contained in prostate cancer-derived EVs promotes the in vitro osteogenic
differentiation of human mesenchymal stromal cells by targeting ARHGAP1 and FAM134A. This in turn
[121]
induces extensive osteoblastic lesions in the bone metastatic microenvironment in vivo .

With regards to breast cancer, we recently demonstrated by in vivo studies the ability of breast cancer-
derived EVs to reach the bone microenvironment and be integrated by osteoblasts and osteoclasts .
[35]
Moreover, these EVs impaired osteoblast viability and differentiation, while enhancing the expression of
pro-osteoclastogenic and inflammatory cytokines. In line with this profile, breast cancer EVs increase
osteoclastogenesis, thus indicating a direct and indirect effect of EVs on osteoclast formation through the
osteoblasts. To complete this picture, the effect of breast cancer cell-EVs on in vitro and in vivo angiogenesis
was also investigated, finding a stimulatory role . Following this paper, Yuan et al. confirmed the crucial
[35]
[134]
role of breast cancer secreted exosomes in establishing a PMN in the bone by stimulating osteoclast
differentiation and function, while, from a mechanistic point of view, they identified the involvement of
exosomal miR-21, which decreases the expression of programmed cell death 4, a factor known to have a
[135]
suppressive function on c-Fos transactivation . The authors also found significantly higher levels of miR-
21 in the serum from breast cancer patients with bone-metastasis compared to that from breast cancer
patients with non-bone metastases, thus giving a prognostic and therapeutic meaning to miR-21 shuttled by
EVs .
[134]
Among other miRNAs driving organotropism of breast cancer cells, there is miR-940. When the human
breast cancer cell line MDA-MB-231, known to induce osteolytic bone metastases, was forced to
overexpress miR-940, it led to the switch towards an osteosclerotic phenotype of bone metastases . A
[121]
similar study, performed by Ye et al. , demonstrated that miR-141-3p shuttled by prostate cancer
[122]
exosomes favors osteoblast differentiation and osteosclerotic bone metastasis development in vivo.

Multiple myeloma is quite frequently associated with a severe impairment of bone integrity, characterized
by the development of extensive osteolytic lesions, caused by an exacerbated osteoclast activity which,