Comparison of Pelvic Landmarks for Leg Length Discrepancy Measurement With Robotic Arm-Assisted Total Hip Arthroplasty.

Background: Leg length discrepancy (LLD) is a common complication after total hip arthroplasty (THA) leading to significant morbidity and dissatisfaction for patients. A popular system for robotic arm-assisted THA utilizes preoperative computed tomography (CT) scans for surgical planning. Accurate measurement of leg length is crucial for restoring appropriate patient anatomy during the procedure. This study investigates the interobserver and interlandmark reliability of 3 different pelvic landmarks for measuring preoperative LLD. Methods: We compiled preoperative pelvic CT scans from 99 robotic arm-assisted THAs for osteoarthritis. Radiologic leg length measurement was performed using the robotic arm-assisted THA application by 2 orthopaedic residents using reference lines bisecting the following pelvic landmarks: the anterior superior iliac spines, acetabular teardrops, and most inferior aspect of the ischial rami. Results: On multivariate analysis, there was no significant difference found (P value = .924) for leg length measurement based on the 3 different pelvic anatomical landmarks. Leg length measurements showed interobserver reliability with significant Pearson correlation coefficients (r = 1.0, 0.94, 0.96, respectively) and nonsignificant differences in LLD means between subjects on paired sample (P value = .158, .085, 0.125, respectively) as well as between landmarks on pairwise comparison. Conclusions: The 3 pelvic landmarks used in this study can be used interchangeably with the lesser trochanter as the femoral reference point to evaluate preoperative LLD on pelvic CT in patients undergoing robotic-arm assisted THA. This study is the first of its kind to evaluate the interobserver and interlandmark reliability of anatomical landmarks on pelvic CT scans and suggests interchangeability of 3 pelvic landmarks for comparing leg length differences.

Acceptance, Intent, Hesitance, and Attitudes Towards SAR-CoV-2 Vaccines Among Healthcare Workers in Michigan, USA.

BACKGROUND: Healthcare workers (HCWs) are critical infrastructure workers for whom COVID-19 vaccination was prioritized. It is believed that healthcare workers would have little or no hesitancy to take the COVID-19 vaccines given the risks of the pandemic to them, their families, and their patients. OBJECTIVE: The study aims to understand the acceptance and attitudes toward COVID-19 vaccines among the HCWs in Michigan. METHODS: A cross-sectional survey was fielded from January 11, 2021, through February 28, 2021. We obtained a representative sample of HCWs at MidMichigan Health. The participants were approximately 1500 clinical and non-clinical HCWs. COVID-19 vaccination acceptance and the intent to be vaccinated were measured with a questionnaire. HCWs indicating hesitance were asked to enter their reasons for hesitance as a free text response. RESULTS: A total of 1,467 HCWs responded to the survey. Overall, 62% indicated they had received both shots; 19.7% reported that they had received the first shot and would take the second; 2.3% noted that they were yet to receive the vaccine but would take both shots; 0.4% reported that they had received the first shot but would not take the second; 5.7% noted that they were unsure; and 9.9% indicated they did not intend to take the vaccine. Factors associated with vaccine hesitance included being female, younger age, having administrative staff or other health workers, having a larger household size, and having received no vaccines in the past year. Vaccine hesitancy concerns included safety, efficacy, antivaccine beliefs, the need for additional information, and a lack of trust. CONCLUSION: This survey revealed that 16% of HCWs in central and northern Michigan were hesitant about COVID-19 vaccines. Vaccine education is needed to increase the acceptance of COVID-19 vaccines among HCWs.

Targeting disseminated estrogen-receptor-positive breast cancer cells in bone marrow.

Estrogen receptor-positive (ER+) breast cancer can recur up to 20 years after initial diagnosis. Delayed recurrences arise from disseminated tumors cells (DTCs) in sites such as bone marrow that remain quiescent during endocrine therapy and subsequently proliferate to produce clinically detectable metastases. Identifying therapies that eliminate DTCs and/or effectively target cells transitioning to proliferation promises to reduce risk of recurrence. To tackle this problem, we utilized a 3D co-culture model incorporating ER+ breast cancer cells and bone marrow mesenchymal stem cells to represent DTCs in a bone marrow niche. 3D co-cultures maintained cancer cells in a quiescent, viable state as measured by both single-cell and population-scale imaging. Single-cell imaging methods for metabolism by fluorescence lifetime (FLIM) of NADH and signaling by kinases Akt and ERK revealed that breast cancer cells utilized oxidative phosphorylation and signaling by Akt to a greater extent both in 3D co-cultures and a mouse model of ER+ breast cancer cells in bone marrow. Using our 3D co-culture model, we discovered that combination therapies targeting oxidative phosphorylation via the thioredoxin reductase (TrxR) inhibitor, D9, and the Akt inhibitor, MK-2206, preferentially eliminated breast cancer cells without altering viability of bone marrow stromal cells. Treatment of mice with disseminated ER+ human breast cancer showed that D9 plus MK-2206 blocked formation of new metastases more effectively than tamoxifen. These data establish an integrated experimental system to investigate DTCs in bone marrow and identify combination therapy against metabolic and kinase targets as a promising approach to effectively target these cells and reduce risk of recurrence in breast cancer.

Enhanced mitochondrial fission suppresses signaling and metastasis in triple-negative breast cancer.

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.

Aim2-mediated/IFN-ß-independent regulation of gastric metaplastic lesions via CD8+ T cells.

Development of gastric cancer is often preceded by chronic inflammation, but the immune cellular mechanisms underlying this process are unclear. Here we demonstrated that an inflammasome molecule, absent in melanoma 2 (Aim2), was upregulated in patients with gastric cancer and in spasmolytic polypeptide-expressing metaplasia of chronically Helicobacter felis-infected stomachs in mice. However, we found that Aim2 was not necessary for inflammasome function during gastritis. In contrast, Aim2 deficiency led to an increase in gastric CD8+ T cell frequency, which exacerbated metaplasia. These gastric CD8+ T cells from Aim2-/- mice were found to have lost their homing receptor expression (sphingosine-1-phosphate receptor 1 [S1PR1] and CD62L), a feature of tissue-resident memory T cells. The process was not mediated by Aim2-dependent regulation of IFN-ß or by dendritic cell-intrinsic Aim2. Rather, Aim2 deficiency contributed to an increased production of CXCL16 by B cells, which could suppress S1PR1 and CD62L in CD8+ T cells. This study describes a potentially novel function of Aim2 that regulates CD8+ T cell infiltration and retention within chronically inflamed solid organ tissue. This function operates independent of the inflammasome, IFN-ß, or dendritic cells. We provide evidence that B cells can contribute to this mechanism via CXCL16.

Plasminogen Activator Inhibitor 1 (PAI1) Promotes Actin Cytoskeleton Reorganization and Glycolytic Metabolism in Triple-Negative 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.

Monitoring implantable immunoisolation devices with intrinsic fluorescence of genipin.

Imaging of implanted hydrogel-based biosystems usually requires indirect labeling of the vehicle or cargo, adding complexity and potential risk of altering functionality. Here, for the first time, it is reported that incorporation of genipin into the design of immunoisolation devices can be harnessed for in vivo imaging. Using cell-compatible in situ cross-linking reactions, a fast, efficient and noncytotoxic procedure is shown to maximize fluorescence of microcapsules. Moreover, genipin is validated as a quantitative imaging probe by injecting increasing doses of microcapsules in the subcutaneous space of mice, obtaining strong, stable fluorescence with good linearity of signal to microcapsule dose over several weeks. This allows immediate assessment of the actual injected dose and monitoring of its position over time, thereby significantly enhancing the efficacy and biosafety of the therapy. These outcomes may facilitate clinical translation and optimize medical applications of multiple hydrogel-based biotechnologies.

Enhanced Bone Metastases in Skeletally Immature Mice.

Bone constitutes the most common site of breast cancer metastases either at time of presentation or recurrent disease years after seemingly successful therapy. Bone metastases cause substantial morbidity, including life-threatening spinal cord compression and hypercalcemia. Given the high prevalence of patients with breast cancer, health-care costs of bone metastases (>$20,000 per episode) impose a tremendous economic burden on society. To investigate mechanisms of bone metastasis, we developed femoral artery injection of cancer cells as a physiologically relevant model of bone metastasis. Comparing young (~6 weeks), skeletally immature mice to old (~6 months) female mice with closed physes (growth plates), we showed significantly greater progression of osteolytic metastases in young animals. Bone destruction increased in the old mice following ovariectomy, emphasizing the pathologic consequences of greater bone turnover and net loss. Despite uniform initial distribution of breast cancer cells throughout the hind limb after femoral artery injection, we observed preferential formation of osteolytic bone metastases in the proximal tibia. Tropism for the proximal tibia arises in part because of TGF-ß, a cytokine abundant in both physes of skeletally immature mice and matrix of bone in mice of all ages. We also showed that age-dependent effects on osteolytic bone metastases did not occur in male mice with disseminated breast cancer cells in bone. These studies establish a model system to specifically focus on pathophysiology and treatment of bone metastases and underscore the need to match biologic variables in the model to relevant subsets of patients with breast cancer.

3D Jet Writing: Functional Microtissues Based on Tessellated Scaffold Architectures.

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.

Functional Isolation of Tumor-Initiating Cells using Microfluidic-Based Migration Identifies Phosphatidylserine Decarboxylase as a Key Regulator.

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.

The effect of mechanosensitive channel MscL expression in cancer cells on 3D confined migration.

Metastatic cancer cells migrate through constricted spaces and experience significant compressive stress, but mechanisms enabling migration in confined geometries remain unclear. Cancer cell migration within confined 3-dimensional (3D) microfluidic channels has been shown to be distinct from 2D cell migration. However, whether 3D confined migration can be manipulated by mechanosensory components has not been examined in detail. In this work, we exogenously introduced a mechanosensitive channel of large conductance (MscL) into metastatic breast cancer cells MDA-MB-231. We discovered that inducing expression of a gain-of-function G22S mutant of MscL in MDA-MB-231 cells significantly reduced spontaneous lung metastasis without affecting the growth of orthotopic tumor implants. To further investigate the effects of G22S MscL on cell migration, we designed a microfluidic device with channels of various cross-sections ranging from a 2D planar environment to narrow 3D constrictions. Both MscL G22S and control breast cancer cells migrated progressively slower in more constricted environments. Migration of cells expressing MscL G22S did not differ from control cells, even though MscL was activated in cells in constricted channels of 3 µm width. Interestingly, we found MscL expressing cells to be more frequently "stuck" at the entrance of the 3 µm channels and failed to migrate into the microchannel. Our work demonstrates the possibility of engineering mechanotransduction for controlling confined cell migration.

A Bifunctional MAPK/PI3K Antagonist for Inhibition of Tumor Growth and Metastasis.

Responses to targeted therapies frequently are brief, with patients relapsing with drug-resistant tumors. For oncogenic MEK and BRAF inhibition, drug resistance commonly occurs through activation of PI3K/AKT/mTOR signaling and immune checkpoint modulation, providing a robust molecular target for concomitant therapy. Here, we evaluated the efficacy of a bifunctional kinase inhibitor (ST-162) that concurrently targets MAPK and PI3K signaling pathways. Treatment with ST-162 produced regression of mutant KRAS- or BRAF-addicted xenograft models of colorectal cancer and melanoma and stasis of BRAF/PTEN-mutant melanomas. Combining ST-162 with immune checkpoint blockers further increased efficacy in a syngeneic KRAS-mutant colorectal cancer model. Nascent transcriptome analysis revealed a unique gene set regulated by ST-162 related to melanoma metastasis. Subsequent mouse studies revealed ST-162 was a potent inhibitor of melanoma metastasis to the liver. These findings highlight the significant potential of a single molecule with multikinase activity to achieve tumor control, overcome resistance, and prevent metastases through modulation of interconnected cell signaling pathways. Mol Cancer Ther; 16(11); 2340-50. ©2017 AACR.