The advent of adaptive manufacturing techniques supports the vision of cell-instructive materials that mimic biological tissues. 3D jet writing, a modified electrospinning process reported herein, yields 3D structures with unprecedented precision and resolution offering customizable pore geometries and scalability to over tens of centimeters. These scaffolds support the 3D expansion and differentiation of human mesenchymal stem cells in vitro. Implantation of these constructs leads to the healing of critical bone defects in vivo without exogenous growth factors. When applied as a metastatic target site in mice, circulating cancer cells home in to the osteogenic environment simulated on 3D jet writing scaffolds, despite implantation in an anatomically abnormal site. Through 3D jet writing, the formation of tessellated microtissues is demonstrated, which serve as a versatile 3D cell culture platform in a range of biomedical applications including regenerative medicine, cancer biology, and stem cell biotechnology.

Background: Mitochondrial dynamics underlies malignant transformation, cancer progression, and response to treatment. Current research presents conflicting evidence for functions of mitochondrial fission and fusion in tumor progression. Here, we investigated how mitochondrial fission and fusion states regulate underlying processes of cancer progression and metastasis in triple-negative breast cancer (TNBC). Methods: We enforced mitochondrial fission and fusion states through chemical or genetic approaches and measured migration and invasion of TNBC cells in 2D and 3D in vitro models. We also utilized kinase translocation reporters (KTRs) to identify single cell effects of mitochondrial state on signaling cascades, PI3K/Akt/mTOR and Ras/ Raf/MEK/ERK, commonly activated in TNBC. Furthermore, we determined effects of fission and fusion states on metastasis, bone destruction, and signaling in mouse models of breast cancer. Results: Enforcing mitochondrial fission through chemical or genetic approaches inhibited migration, invasion, and metastasis in TNBC. Breast cancer cells with predominantly fissioned mitochondria exhibited reduced activation of Akt and ERK both in vitro and in mouse models of breast cancer. Treatment with leflunomide, a potent activator of mitochondrial fusion proteins, overcame inhibitory effects of fission on migration, signaling, and metastasis. Mining existing datasets for breast cancer revealed that increased expression of genes associated with mitochondrial fission correlated with improved survival in human breast cancer. Conclusions: In TNBC, mitochondrial fission inhibits cellular processes and signaling pathways associated with cancer progression and metastasis. These data suggest that therapies driving mitochondrial fission may benefit patients with breast cancer.

Migration and invasion of cancer cells constitute fundamental processes in tumor progression and metastasis. Migratory cancer cells commonly upregulate expression of plasminogen activator inhibitor 1 (PAI1), and PAI1 correlates with poor prognosis in breast cancer. However, mechanisms by which PAI1 promotes migration of cancer cells remain incompletely defined. Here we show that increased PAI1 drives rearrangement of the actin cytoskeleton, mitochondrial fragmentation, and glycolytic metabolism in triple-negative breast cancer (TNBC) cells. In two-dimensional environments, both stable expression of PAI1 and treatment with recombinant PAI1 increased migration, which could be blocked with the specific inhibitor tiplaxtinin. PAI1 also promoted invasion into the extracellular matrix from coculture spheroids with human mammary fibroblasts in fibrin gels. Elevated cellular PAI1 enhanced cytoskeletal features associated with migration, actin-rich migratory structures, and reduced actin stress fibers. In orthotopic tumor xenografts, we discovered that TNBC cells with elevated PAI1 show collagen fibers aligned perpendicular to the tumor margin, an established marker of invasive breast tumors. Further studies revealed that PAI1 activates ERK signaling, a central regulator of motility, and promotes mitochondrial fragmentation. Consistent with known effects of mitochondrial fragmentation on metabolism, fluorescence lifetime imaging microscopy of endogenous NADH showed that PAI1 promotes glycolysis in cell-based assays, orthotopic tumor xenografts, and lung metastases. Together, these data demonstrate for the first time that PAI1 regulates cancer cell metabolism and suggest targeting metabolism to block motility and tumor progression.Implications: We identified a novel mechanism through which cancer cells alter their metabolism to promote tumor progression.

Isolation of tumor-initiating cells currently relies on markers that do not reflect essential biologic functions of these cells. We proposed to overcome this limitation by isolating tumor-initiating cells based on enhanced migration, a function tightly linked to tumor-initiating potential through epithelial-to-mesenchymal transition (EMT). We developed a high-throughput microfluidic migration platform with automated cell tracking software and facile recovery of cells for downstream functional and genetic analyses. Using this device, we isolated a small subpopulation of migratory cells with significantly greater tumor formation and metastasis in mouse models. Whole transcriptome sequencing of migratory versus non-migratory cells from two metastatic breast cancer cell lines revealed a unique set of genes as key regulators of tumor-initiating cells. We focused on phosphatidylserine decarboxylase (PISD), a gene downregulated by 8-fold in migratory cells. Breast cancer cells overexpressing PISD exhibited reduced tumor-initiating potential in a high-throughput microfluidic mammosphere device and mouse xenograft model. PISD regulated multiple aspects of mitochondria, highlighting mitochondrial functions as therapeutic targets against cancer stem cells. This research establishes not only a novel microfluidic technology for functional isolation of tumor-initiating cells regardless of cancer type, but also a new approach to identify essential regulators of these cells as targets for drug development.

Aim2 deletion exacerbates SPEM. We identified Aim2 in a screen of upregulated gastric genes in 2 mouse models of SPEM: (a) chronic (6-month) H. felis infection and (b) gastric tissue-specific overexpression of IFN-γ (H + / K +-ATPase-IFN-γ) (18) (Figure 1A). The upregulation of Aim2 in gastric tissue of chronically H. felis-infected mice at 6 months was corroborated by real-time quantitative PCR (RT-qPCR) (Figure 1B). To study the role of Aim2 in SPEM, we used mice deficient in Aim2 (B6.129P2-Aim2 Gt(CSG445)Byg /J; Aim2-/-) and chronically infected them with H. felis. We confirmed that Aim2 deletion abrogated Aim2 expression in the stomach (Figure 1B). Aim2 deficiency worsened gastric immunopathology (Figure 1C), increased stomach weight (Figure 1D), exacerbated SPEM (Figure 1, E and F), and enhanced parietal cell atrophy (Figure 1G). All the latter characteristics epitomize features of enhanced gastric preneoplastic development in Aim2-/mice. Aim2 lacks a significant effect on gastric inflammasome activity. Because Aim2 functions as a component of the inflammasome (13-15), we investigated whether Aim2 was mediating its observed effects by regulating IL-1β and IL-18 secretion. We found gastric explants from 6-month infected Aim2-/stomachs did not show a significant effect on the secretion of IL-1β or IL-18 (Supplemental Figure 1, A and B; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.94035DS1). Hence, these observations did not provide mechanistic insight into how the loss of Aim2 led to increased SPEM. This prompted us to dissect the mechanism of how Aim2 inhibits SPEM further. Aim2 is not required for the expression of the inhibitory Fcγ receptor. Because a previous report showed that Aim2 is necessary for the expression of the inhibitory Fcγ receptor (FcγRIIB) (19), we investigated a possible alternative mechanism for Aim2 via this protein. FcγRIIB is a lupus susceptibility protein that suppresses antibody production in B cells (20) and blocks maturation of dendritic cells (21). However, we did not observe a significant effect of Aim2 deficiency on FcγRIIB expression in the inflamed stomach (Supplemental Figure 1C). Aim2 deficiency increases gastric CD8 + T cell frequency in the chronically inflamed stomach. Because the mechanism of Aim2 in gastric pathology remained unclear, we screened several gastric immune populations from chronically infected Aim2-/stomachs versus WT stomachs. Previous reports have documented several immune subtypes that increase during chronic gastric inflammation and SPEM (22-28). These include CD11b + Ly6G + myeloid-derived suppressor cells (MDSCs) (22-24), CD11b + Ly6Gmyeloid cells (22), CD4 + and CD8 + T cells (25-27), and B220 + IgM + B cells (28). We therefore screened these populations in our system (Figure 2A). In chronically infected WT stomachs, we observed a significant increase in all these populations relative to uninfected WT stomachs (Figure 2A). However, gastric CD8 + T cells were the only population whose frequency was affe...