Angiocrine signals regulate quiescence and therapy resistance in bone metastasis
Amit Singh, Vimal Veeriah, Pengjun Xi, Rossella Labella, Junyu Chen, Sara G. Romeo, Saravana K. Ramasamy, Anjali P. Kusumbe
Amit Singh, Vimal Veeriah, Pengjun Xi, Rossella Labella, Junyu Chen, Sara G. Romeo, Saravana K. Ramasamy, Anjali P. Kusumbe
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Research Article Bone biology Vascular biology

Angiocrine signals regulate quiescence and therapy resistance in bone metastasis

Abstract

Bone provides supportive microenvironments for hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) and is a frequent site of metastasis. While incidences of bone metastases increase with age, the properties of the bone marrow microenvironment that regulate dormancy and reactivation of disseminated tumor cells (DTCs) remain poorly understood. Here, we elucidate the age-associated changes in the bone secretome that trigger proliferation of HSCs, MSCs, and DTCs in the aging bone marrow microenvironment. Remarkably, a bone-specific mechanism involving expansion of pericytes and induction of quiescence-promoting secretome rendered this proliferative microenvironment resistant to radiation and chemotherapy. This bone-specific expansion of pericytes was triggered by an increase in PDGF signaling via remodeling of specialized type H blood vessels in response to therapy. The decline in bone marrow pericytes upon aging provides an explanation for loss of quiescence and expansion of cancer cells in the aged bone marrow microenvironment. Manipulation of blood flow — specifically, reduced blood flow — inhibited pericyte expansion, regulated endothelial PDGF-B expression, and rendered bone metastatic cancer cells susceptible to radiation and chemotherapy. Thus, our study provides a framework to recognize bone marrow vascular niches in age-associated increases in metastasis and to target angiocrine signals in therapeutic strategies to manage bone metastasis.

Authors

Amit Singh, Vimal Veeriah, Pengjun Xi, Rossella Labella, Junyu Chen, Sara G. Romeo, Saravana K. Ramasamy, Anjali P. Kusumbe

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Figure 4

Quiescence-promoting secreted factors decline in the aged BM microenvironment.

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Quiescence-promoting secreted factors decline in the aged BM microenviro...
(A) FACS quantification of proliferating MDA-MB-231-GFP cells in culture by Ki-67 immunostaining in PBS and aged BM secretome–treated (aged-BM-sec) cells. Data represent mean ± SD (n = 5 replicates); 2-tailed unpaired t test. (B) Bioluminescence analysis of sham (PBS) and aged-BM-sec injected in young mice tibiae along with cancer cells. Data represent mean ± SD (n = 5 replicates); 2-tailed unpaired t test. (C) FACS quantification of the frequency of MDA-MB-231-GFP cells in single-cell suspensions of tibiae from sham- (PBS-), young-BM-sec– (secretome from young 5-week-old mice), and aged-BM-sec–injected (secretome from aged 70-week-old mice) groups. Data represent mean ± SD (n = 5 replicates); 1-way ANOVA by Dunnett’s multiple-comparisons test. (D) FACS quantification of Ki-67+ MDA-MB-231-GFP cells in sham- (PBS-), young-BM-sec–, and aged-BM-sec–injected tibiae from young mice. Data represent mean ± SD (n = 6 replicates); 1-way ANOVA by Dunnett’s multiple-comparisons test. (E) FACS quantifications of the quiescent (G0) MDA-MB-231-GFP cells in young tibia, based on Hoechst and Pyronin Y staining of single-cell suspensions of the tibiae. Data represent mean ± SD (n = 6 replicates); 1-way ANOVA by Dunnett’s multiple-comparisons test. (F) The heatmap shows the most significantly differentially regulated secreted factors involved in maintaining stem and cancer cell quiescence. Color intensity indicates row-scaled-normalized log2(cpm) expression values. (G) qPCR analysis of Bmp4, Bmp6, Bmp7, Kitl, Tgfb2, and Thbs2 expression (normalized to Actb) by aged tibiae relative to young (Rel. fold mRNA exp.). Data represent mean ± SD (n = 6 replicates); 2-tailed unpaired t tests. (H) Representative FACS contour plots show Hoechst and Pyronin Y staining on gated GFP+ MDA-MB-231 cells in single-cell suspensions of radiation-treated and control aged mouse tibiae that were injected with MDA-MB-231-GFP cells 2 weeks after intratibial injections. The left graph shows quantification of quiescent MDA-MB-231-GFP cells in radiation-treated (IRR) aged mice. Data represent mean ± SD (n = 6 replicates); 2-tailed unpaired t tests. The right graph shows quantification of quiescent MDA-MB-231-GFP cells in radiation-treated young mice. Data represent mean ± SD (n = 5 replicates); 2-tailed unpaired t tests. **P < 0.01, ***P < 0.001, ****P < 0.0001.