miR-100 and miR-125b Contribute to Enhanced 3D Growth and Invasiveness and can be Functionally Transferred to Silence Target Genes in Recipient Cells.

Extracellular communication via the transfer of vesicles and nanoparticles is now recognized to play an important role in tumor microenvironment interactions. Cancer cells upregulate and secrete abundant levels of miR-100 and miR-125b that can alter gene expression by both cell- and non-cell-autonomous mechanisms. We previously showed that these miRNAs activate Wnt signaling in colorectal cancer (CRC) through noncanonical pairing with 5 negative regulators of Wnt signaling. To identify additional targets of miR-100 and miR-125b , we used bioinformatic approaches comparing multiple CRC cell lines, including knockout lines lacking one or both of these miRNAs. From an initial list of 96 potential mRNA targets, we tested 15 targets with 8 showing significant downregulation in the presence of miR-100 and miR-125b . Among these, Cingulin (CGN) and Protein tyrosine phosphatase receptor type-R (PTPRR) are downregulated in multiple cancers, consistent with regulation by increased levels of miR-100 and miR-125b. We also show that increased cellular levels of miR-100 and miR-125b enhance 3D growth and invasiveness in CRC and glioblastoma cell lines. Lastly, we demonstrate that extracellular transfer of miR-100 and miR-125b can silence both reporter and endogenous mRNA targets in recipient cells and also increase the invasiveness of recipient spheroid colonies when grown under 3D conditions in type I collagen.

Mammary tissue-derived extracellular matrix hydrogels reveal the role of irradiation in driving a pro-tumor and immunosuppressive microenvironment.

Radiation therapy (RT) is essential for triple negative breast cancer (TNBC) treatment. However, patients with TNBC continue to experience recurrence after RT. The role of the extracellular matrix (ECM) of irradiated breast tissue in tumor recurrence is still unknown. In this study, we evaluated the structure, molecular composition, and mechanical properties of irradiated murine mammary fat pads (MFPs) and developed ECM hydrogels from decellularized tissues (dECM) to assess the effects of RT-induced ECM changes on breast cancer cell behavior. Irradiated MFPs were characterized by increased ECM deposition and fiber density compared to unirradiated controls, which may provide a platform for cell invasion and proliferation. ECM component changes in collagens I, IV, and VI, and fibronectin were observed following irradiation in both MFPs and dECM hydrogels. Encapsulated TNBC cell proliferation and invasive capacity was enhanced in irradiated dECM hydrogels. In addition, TNBC cells co-cultured with macrophages in irradiated dECM hydrogels induced M2 macrophage polarization and exhibited further increases in proliferation. Our study establishes that the ECM in radiation-damaged sites promotes TNBC invasion and proliferation as well as an immunosuppressive microenvironment. This work represents an important step toward elucidating how changes in the ECM after RT contribute to breast cancer recurrence.

Development of an alginate-Matrigel hydrogel system to evaluate cancer cell behavior in the stiffness range of the bone marrow.

Bone metastasis is highly prevalent in breast cancer patients with metastatic disease. These metastatic cells may eventually form osteolytic lesions and affect the integrity of the bone, causing pathological fractures and impairing patient quality of life. Although some mechanisms have been determined in the metastatic cascade to the bone, little is known about how the mechanical cues of the bone marrow microenvironment influence tumor cell growth and invasion once they have homed to the secondary site. The mechanical properties within the bone marrow range from 0.5 kPa in the sinusoidal region to 40 kPa in the endosteal region. Here, we report an alginate-Matrigel hydrogel that can be modulated to the stiffness range of the bone marrow and used to evaluate tumor cell behavior. We fabricated alginate-Matrigel hydrogels with varying calcium sulfate (CaSO4) concentrations to tune stiffness, and we demonstrated that these hydrogels recapitulated the mechanical properties observed in the bone marrow microenvironment (0.7-16 kPa). We encapsulated multiple breast cancer cell lines into these hydrogels to assess growth and invasion. Tumor cells in stiffer hydrogels exhibited increased proliferation and enhanced elongation compared to lower stiffness hydrogels, which suggests that stiffer environments in the bone marrow promote cellular invasive capacity. This work establishes a system that replicates bone marrow mechanical properties to elucidate the physical factors that contribute to metastatic growth.