Glen L. Niebur

Mammary gland development requires coordinated signaling between epithelial and stromal compartments. While CXC chemokine receptor 2 (CXCR2) is known to regulate inflammatory and tissue remodeling processes in disease, including breast cancer, its role in mammary gland morphogenesis is not well understood. Here, we examined postnatal mammary gland development in germline Cxcr2 knockout mice. Loss of Cxcr2 resulted in delayed ductal outgrowth, reduced epithelial area, and altered stromal architecture, including decreased adipocyte size and increased collagen deposition, despite preserved branching organization. These defects occurred in the context of severe systemic immune dysregulation, marked by expansion of granulocytic populations and loss of adaptive immune cells. Together, our findings indicate that CXCR2 indirectly regulates mammary gland development through coordination between the epithelium and stroma, rather than acting through epithelial cell-intrinsic mechanisms. Because the same myeloid expansion, immune suppression, and collagen-rich stromal remodeling that CXCR2 controls during normal development are hallmarks of the pro-tumorigenic breast microenvironment, these findings establish CXCR2 as a regulator of the immune and stromal landscape of the mammary gland and provide a developmental framework for understanding its contributions to the breast tumor microenvironment.

Osteoblastic bone metastasis, in which disseminated tumor cells drive net bone formation, is a clinically distinct and mechanistically underexplored form of skeletal disease that is enriched in hormone receptor-positive breast cancers. Preclinical models of bone metastasis from breast cancer predominantly rely on immunodeficient hosts inoculated with osteolytic human breast cancer cell lines, limiting the study of immune-dependent mechanisms of bone remodeling. Here we describe the development and characterization of an immunocompetent, syngeneic osteoblastic bone metastasis model using intratibial injection of PyMT-CK(OB), a luciferase-expressing derivative of the MMTV-PyMT mammary carcinoma cell line, in FVB/N mice. PyMT-CK(OB) cells produced detectable bioluminescent signal after intratibial injection, enabling longitudinal monitoring of tumor progression. Micro-computed tomography (µCT) revealed significant increases in trabecular bone volume fraction and trabecular number at three and four weeks post-injection, consistent with osteoblastic remodeling. Histological analysis confirmed dense bone lesion formation in tumor-bearing bones. Critically, this osteoblastic phenotype was entirely absent in immunodeficient NOD SCID hosts, despite robust tumor growth, supporting a role for immune competence in tumor-induced bone formation. Loss of bioluminescent signal in immunocompetent mice reflected either immune pressure on reporter gene expression or limited space for cancer cell expansion in the bone, rather than tumor regression or hypoxia, as confirmed by hypoxia imaging and histological endpoint analysis. In contrast, a second PyMT cell subline, PyMT-CF, maintained sustained bioluminescent signal and produced predominantly osteolytic lesions, providing a complementary syngeneic model of osteolytic disease from the same parental background. In vitro hydrogel coculture experiments and protein array analysis of conditioned media revealed that the PyMT sublines have differing impact on MC3T3 osteoblast mineralization, identifying candidate mediators of divergent bone remodeling phenotypes. R7 mammary carcinoma cells derived from MMTV-RON transgenic mouse mammary tumors did not induce measurable bone remodeling under equivalent experimental conditions. Together, these models provide a validated, immunologically intact framework for studying the mechanistic basis of osteoblastic bone metastasis and evaluating therapeutic interventions targeting the tumor-bone microenvironment.

Bone provides a favourable niche for breast cancer colonization and metastatic progression. Breast cancer cells are attracted to the bone microenvironment, where they induce bone cells to resorb bone, which enhances tumour cell proliferation in a positive feedback loop often referred to as the vicious cycle. While this phenomenon is established, the molecular interactions between cancer cells and bone cells are not well defined. CXCL5/CXCR2 signalling has recently been shown to promote breast cancer colonization to the bone. Here, we investigate the effects of osteoblasts and osteocytes on breast cancer cell proliferation in engineered two-dimensional (2D) and three-dimensional (3D) models. We observed that osteoblasts and osteocytes induce proliferative effects on cancer cells. Specifically, bone cells increase cancer proliferation in 2D culture, and osteoblasts increase cancer growth more than osteocytes in 3D models. Moreover, osteocyte interaction with cancer cells in 3D models is stiffness dependent. We show that these effects depend on the CXCL5/CXCR2 signalling axis. Taken together, we demonstrate that osteoblasts enhance cancer growth in a bone metastatic niche and that this effect is reversible with CXCL5/CXCR2 inhibition.

Bone provides a favorable niche for breast cancer colonization and metastatic progression. Breast cancer cells are attracted to the bone microenvironment where they induce bone cells to resorb bone, which enhances tumor cell proliferation in a positive feedback loop often referred to as the vicious cycle. While this phenomenon is established, the molecular interactions between cancer cells and bone cells are not well defined. CXCL5/CXCR2 signaling has recently been shown to promote breast cancer colonization to the bone. Here, we investigate the effects of osteoblasts and osteocytes on breast cancer cell proliferation in engineered two- and three-dimensional models. We observed that osteoblasts and osteocytes induce proliferative effects on cancer cells. Specifically, bone cells increase cancer proliferation in 2D culture and osteoblasts increase cancer growth more than osteocytes in 3D models. Moreover, osteocyte interaction with cancer cells in 3D models are stiffness dependent. We show that these effects depend on the CXCL5/CXCR2 signaling axis. Taken together, we demonstrate that osteoblasts drive cancer growth in a bone metastatic niche and that this effect can be rescued with CXCL5/CXCR2 inhibition.

One factor that could affect the success of bone ingrowth in total knee replacements is the mechanical environment at the bone-implant interface. We applied a mechanoregulatory model to predict the evolution of tissue formation and ingrowth for two scaffold porosities and a range of loads. When the compressive motion was applied across the gap, low levels of shear displacement improved bone formation by transmitting strain deeper into the scaffold. Ingrowth scaffolds with higher porosity are more tolerant to shear micromotion because their compliance leads to a mechanical environment that promotes tissue differentiation into the scaffold, resulting in greater interface stiffness.

Osteocytes regulate the response of osteoclasts and osteoblasts to mechanical loading through signaling molecules, the levels of which are controlled by post-translational modification or degradation and by differential gene transcription and translation. The magnitude and mode of bone tissue deformation that elicits a transcriptional response in individual osteocytes in situ has been difficult to quantify. We measured SOST, Wnt11, TNF, and FRZB gene expression in osteocytes within loaded and unloaded control porcine trabecular bone explants using RNAScope® and compared the local tissue level strain and strain gradient—which we used as an indicator of potential poroelastic fluid flow—in the tissue surrounding osteocytes with high vs. low gene expression. The measured expression of all four genes differed between loaded and unloaded explants, on average, with the mean SOST expression level decreasing by 45%. In the loaded explants, gene expression was altered from baseline in about 30% of the osteocytes, and they were surrounded by tissue with higher strain and strain gradient than the 20 to 25% of osteocytes that remained near baseline expression. Both deviatoric strain and hydrostatic strain gradient were sensitive and specific predictors of the mechanobiological response for individual genes as well as combinations. SOST expression was highly related to elevated strain gradient, providing evidence that osteocytes respond to fluid flow in the lacunar-canalicular system.

We show that MDA-MB-231 cells migrate to trabecular bone explants with intact marrow faster than to bone devoid of marrow or devitalized bone. Cells also migrated toward bone with intact marrow when cultured in three-dimensional gelatin constructs. They were more likely to migrate in stiff versus soft substrates, but their speed was not affected by stiffness.