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STUDY QUESTION Can artificial intelligence (AI) algorithms developed to assist embryologists in evaluating embryo morphokinetics be enriched with multi-centric clinical data to better predict clinical pregnancy outcome? SUMMARY ANSWER Training algorithms on multi-centric clinical data significantly increased AUC compared to algorithms that only analyzed the time-lapse system (TLS) videos. WHAT IS KNOWN ALREADY Several AI-based algorithms have been developed to predict pregnancy, most of them based only on analysis of the time-lapse recording of embryo development. It remains unclear, however, whether considering numerous clinical features can improve the predictive performances of time-lapse based embryo evaluation. STUDY DESIGN, SIZE, DURATION A dataset of 9986 embryos (95.60% known clinical pregnancy outcome, 32.47% frozen transfers) from 5226 patients from 14 European fertility centers (in two countries) recorded with three different TLS was used to train and validate the algorithms. A total of 31 clinical factors were collected. A separate test set (447 videos) was used to compare performances between embryologists and the algorithm. PARTICIPANTS/MATERIALS, SETTING, METHODS Clinical pregnancy (defined as a pregnancy leading to a fetal heartbeat) outcome was first predicted using a 3D convolutional neural network that analyzed videos of the embryonic development up to 2 or 3 days of development (33% of the database) or up to 5 or 6 days of development (67% of the database). The output video score was then fed as input alongside clinical features to a gradient boosting algorithm that generated a second score corresponding to the hybrid model. AUC was computed across 7-fold of the validation dataset for both models. These predictions were compared to those of 13 senior embryologists made on the test dataset. MAIN RESULTS AND THE ROLE OF CHANCE The average AUC of the hybrid model across all 7-fold was significantly higher than that of the video model (0.727 versus 0.684, respectively, P = 0.015; Wilcoxon test). A SHapley Additive exPlanations (SHAP) analysis of the hybrid model showed that the six first most important features to predict pregnancy were morphokinetics of the embryo (video score), oocyte age, total gonadotrophin dose intake, number of embryos generated, number of oocytes retrieved, and endometrium thickness. The hybrid model was shown to be superior to embryologists with respect to different metrics, including the balanced accuracy (P ≤ 0.003; Wilcoxon test). The likelihood of pregnancy was linearly linked to the hybrid score, with increasing odds ratio (maximum P-value = 0.001), demonstrating the ranking capacity of the model. Training individual hybrid models did not improve predictive performance. A clinic hold-out experiment was conducted and resulted in AUCs ranging between 0.63 and 0.73. Performance of the hybrid model did not vary between TLS or between subgroups of embryos transferred at different days of embryonic development. The hybrid model did fare better for patients older than 35 years (P LIMITATIONS, REASONS FOR CAUTION Participant centers were located in two countries, thus limiting the generalization of our conclusion to wider subpopulations of patients. Not all clinical features were available for all embryos, thus limiting the performances of the hybrid model in some instances. WIDER IMPLICATIONS OF THE FINDINGS Our study suggests that considering clinical data improves pregnancy predictive performances and that there is no need to retrain algorithms at the clinic level unless they follow strikingly different practices. This study characterizes a versatile AI algorithm with similar performance on different time-lapse microscopes and on embryos transferred at different development stages. It can also help with patients of different ages and protocols used but with varying performances, presumably because the task of predicting fetal heartbeat becomes more or less hard depending on the clinical context. This AI model can be made widely available and can help embryologists in a wide range of clinical scenarios to standardize their practices. STUDY FUNDING/COMPETING INTEREST(S) Funding for the study was provided by ImVitro with grant funding received in part from BPIFrance (Bourse French Tech Emergence (DOS0106572/00), Paris Innovation Amorçage (DOS0132841/00), and Aide au Développement DeepTech (DOS0152872/00)). A.B.-C. is a co-owner of, and holds stocks in, ImVitro SAS. A.B.-C. and F.D.M. hold a patent for ‘Devices and processes for machine learning prediction of in vitro fertilization’ (EP20305914.2). A.D., N.D., M.M.F., and F.D.M. are or have been employees of ImVitro and have been granted stock options. X.P.-V. has been paid as a consultant to ImVitro and has been granted stocks options of ImVitro. L.C.-D. and C.G.-S. have undertaken paid consultancy for ImVitro SAS. The remaining authors have no conflicts to declare. TRIAL REGISTRATION NUMBER N/A.

This file contains figures in the supplementary information. These data complements data in the main figures. It contains: Figure S1: Microfluidic Assay for cell migration in 3D ECM; Figure S2: Macrophages increase cancer cell invasion rate in 3D ECM; macrophages increase cancer cell migration total speed but decrease directedness on 2D substrates; Figure S3: MMP activity is crucial for cancer cell migration, and macrophages enhance cancer cell MMP expression; Figure S4: The abilities of macrophages to enhance cancer cell migration dynamics and the expression of MMPs are due primarily to the paracrine factors secreted by macrophages; Figure S5: Neutralizing antibody blocking is specific to factors released by macrophages; Figure S6: Blocking TNFalpha and TGFbeta1 secreted by macrophages significantly reduced macrophage-induced MMP expression and macrophage-enhanced cancer cell invasion rate; Figure S7: TNFalpha and TGFbeta1 secreted by macrophages mostly accounts for macrophage-enhanced cancer cell migration total speed and directedness; Figure S8: TGFbeta1 has a larger effect on cancer cell migration total speed than TNFalpha. TNFalpha and TGFbeta1 synergistically increase cancer cell migration directedness; Figure S9: Cancer cell migration total speed is correlated with cancer cell expression of MT1-MMP, while cancer cell migration directedness is correlated with cancer cell expression of MMP1; Figure S10: Inhibiting both TNFalpha and TGFbeta1 in a mice model of breast tumor effectively reduces lung metastasis; Figure S11: TNFalpha and TGFbeta1 co-treatment synergistically up-regulate cancer cell MMP1 protein production and secretion. TGFbeta1 is mainly responsible for the increase in MT1-MMP protein production; Figure S12: TNFalpha and TGFbeta1 co-treatment synergistically enhance NF-κB nuclear localization in MDA-MB-435S cells. Fig. S1 and S2 is related to Fig. 1; Fig. S3 and S4 is related to Fig. 2; Fig. S5, S6, S7 is related to Fig. 3; Fig. S8-11 is related to Fig. 4; Fig. S12 is related to Fig. 6

This files contains detailed methods on procedures used in this study, which complements the method described in the main paper. It also expands on the methods described in the main paper. Specifically, it contains method on Microfluidic 3D cell migration assay; 2D Cell Migration Assay; Immunofluorescent staining, confocal microscopy, and DQ-Collagen I Release Assay; ELISA; Cancer cell-macrophage co-culture for western blot analysis; Treatment of cancer cells for western blot analysis; Protein isolation and western blot analysis; Total RNA extraction and mRNA detection; In vivo metastasis assay

The ability of a cancer cell to migrate through the dense extracellular matrix within and surrounding the solid tumor is a critical determinant of metastasis. Macrophages enhance invasion and metastasis in the tumor microenvironment, but the basis for their effects is not fully understood. Using a microfluidic 3D cell migration assay, we found that the presence of macrophages enhanced the speed and persistence of cancer cell migration through a 3D extracellular matrix in a matrix metalloproteinases (MMP)-dependent fashion. Mechanistic investigations revealed that macrophage-released TNFα and TGFβ1 mediated the observed behaviors by two distinct pathways. These factors synergistically enhanced migration persistence through a synergistic induction of NF-κB–dependent MMP1 expression in cancer cells. In contrast, macrophage-released TGFβ1 enhanced migration speed primarily by inducing MT1-MMP expression. Taken together, our results reveal new insights into how macrophages enhance cancer cell metastasis, and they identify TNFα and TGFβ1 dual blockade as an antimetastatic strategy in solid tumors. Cancer Res; 77(2); 279–90. ©2016 AACR.

This file contains the figure captions for the supplementary information figures. Again, Fig. S1 and S2 is related to Fig. 1; Fig. S3 and S4 is related to Fig. 2; Fig. S5, S6, S7 is related to Fig. 3; Fig. S8-11 is related to Fig. 4; Fig. S12 is related to Fig. 6. Each figure caption describes its corresponding figure, summarize the results, and describes the statistics of the data.

Study question Can AI algorithms assist embryologists in evaluating embryos from any time-lapse system (TLS) along with clinical data to better predict pregnancy outcomes and reduce time-to-pregnancy? Summary answer Our algorithm (Embryoly) significantly increases accuracy in predicting clinical pregnancy by 26.9% amongst embryos deemed of fair and good quality when clinical data is included. What is known already Embryologists routinely use defined morpho-kinetic criteria to decide which embryo to transfer, and yet, many embryos deemed of good quality fail to lead to a pregnancy. Thus, AI algorithms to assist embryologists in objectively selecting the most promising embryos are in demand. To date, several reports indicate that AI algorithms are capable of predicting pregnancy clinical outcomes but to the best of our knowledge they only consider visual data (or together with a small set of clinical features) from individual TLI systems to generate their predictions. Study design, size, duration A dataset of 6790 embryos (97.82% known clinical pregnancy outcome, 31.47% frozen transfers) from 2519 patients from 11 European fertility centers recorded with 4 different TLS (GERI-Merck, Embryoscope & EmbryoscopePlus-Vitrolife and MIRI-Esco) was used to train and validate Embryoly. Nine out of 93 clinical factors were identified as being the most predictive, including woman age, woman and man BMI and AMH levels. Performances were evaluated on a separate test dataset (393 videos). Participants/materials, setting, methods Clinical pregnancy outcome was predicted using a 3D convolutional neural network that analyzed up to 5 days of embryo development. The output score was further analyzed considering the clinical features to generate a second clinical score. Both predictions were compared to those of 10 senior embryologists made on the same test dataset (with and without clinical features). Embryo quality was assessed as: poor, fair, good. Unless specified otherwise, McNemar test was used for statistical tests. Main results and the role of chance Overall accuracy of embryologists in predicting clinical pregnancy based on videos alone was 57.25% (CI 95% : 52.34% - 62.16%) compared to 60.56% (CI 95% : 55.71% - 65.41%) for Embryoly (p = 0.35). When videos were analyzed together with the clinical factors, overall accuracy of embryologists was significantly lower than Embryoly (60.05% [CI 95% : 55.19% - 64.91%] vs 68.19% [CI 95% : 63.57% - 72.82%], p-value=0.015, respectively). Clinical factors significantly increased our accuracy by 7.63% (p-value=0.030). More specifically, Embryoly algorithms fared better in terms of detecting false positives (31.30% vs 19.34%) compared to embryologists, with a specificity of 74.4% vs. 58.6%, respectively. If we consider only embryos of fair and good quality (71.50% of our test dataset) Embryoly’s accuracy was 13.52% higher than that of embryologists. This translates into AI having an even better ability to detect false positives for embryos that could be seen as good candidates for transfer (20.28% false positives against 42.70% for the embryologists). Embryoly performs differently across selected TLS when analyzing videos alone, but not when clinical data was also considered (chi2 test, p Limitations, reasons for caution As of today, Embryoly’s accuracy in predicting the outcome of poor-quality embryos is not different to that of embryologists (79.46% vs 84.96%; p-value=0.19). We are improving this by exposing Embryoly to more “poor quality” embryos, so as to also identify poor quality embryos with unexpected potential for implantation. Wider implications of the findings Our pioneering findings support the use of AI for a standardized and couple-centered care in clinical embryology, integrating male and female factors with embryo development analyses from multiple TLS. Our approach has the potential to cost-effectively reduce time to pregnancy and is another step toward a personalized embryo transfer strategy. Trial registration number Not applicable

Background Understanding the molecular mechanisms of metastatic dissemination, the leading cause of death in cancer patients, is required to develop novel, effective therapies. Extravasation, an essential rate-limiting process in the metastatic cascade, includes three tightly coordinated steps: cancer cell adhesion to the endothelium, trans -endothelial migration, and early invasion into the secondary site. Focal adhesion proteins, including Tln1 and FAK, regulate the cytoskeleton dynamics: dysregulation of these proteins is often associated with metastatic progression and poor prognosis. Methods Here, we studied the previously unexplored role of these targets in each extravasation step using engineered 3D in vitro models, which recapitulate the physiological vascular niche experienced by cancer cells during hematogenous metastasis. Results Human breast cancer and fibrosarcoma cell lines respond to Cdk5/Tln1/FAK axis perturbation, impairing their metastatic potential. Vascular breaching requires actin polymerization-dependent invadopodia formation. Invadopodia generation requires the structural function of FAK and Tln1 rather than their activation through phosphorylation. Our data support that the inhibition of FAKS732 phosphorylation delocalizes ERK from the nucleus, decreasing ERK phosphorylated form. These findings indicate the critical role of these proteins in driving trans-endothelial migration. In fact, both knock-down experiments and chemical inhibition of FAK dramatically reduces lung colonization in vivo and TEM in microfluidic setting. Altogether, these data indicate that engineered 3D in vitro models coupled to in vivo models, genetic, biochemical, and imaging tools represent a powerful weapon to increase our understanding of metastatic progression. Conclusions These findings point to the need for further analyses of previously overlooked phosphorylation sites of FAK, such as the serine 732, and foster the development of new effective antimetastatic treatments targeting late events of the metastatic cascade.

Purpose: Conventional wisdom posits that aqueous humor leaves the eye by passive bulk flow without involving energy-dependent processes. However, recent studies have shown that active processes, such as cell contractility, contribute to outflow regulation. Here, we examine whether inhibiting cellular metabolism affects outflow facility in mice. Methods: We measured outflow facility in paired enucleated eyes from C57BL/6J mice using iPerfusion. We had three Experimental Sets: ES1, perfused at 35°C versus 22°C; ES2, perfused with metabolic inhibitors versus vehicle at 35°C; and ES3, perfused at 35°C versus 22°C in the presence of metabolic inhibitors. Inhibitors targeted glycolysis and oxidative phosphorylation (2-deoxy-D-glucose, 3PO and sodium azide). We also measured adenosine triphosphate (ATP) levels in separate murine anterior segments treated like ES1 and ES2. Results: Reducing temperature decreased facility by 63% [38%, 78%] (mean [95% confidence interval (CI)], n = 10 pairs; P = 0.002) in ES1 after correcting for changes in viscosity. Metabolic inhibitors reduced facility by 21% [9%, 31%] (n = 9, P = 0.006) in ES2. In the presence of inhibitors, temperature reduction decreased facility by 44% [29%, 56%] (n = 8, P < 0.001) in ES3. Metabolic inhibitors reduced anterior segment adenosine triphosphate (ATP) levels by 90% [83%, 97%] (n = 5, P

Understanding the molecular mechanisms of metastatic dissemination, the leading cause of death in cancer patients, is required to develop novel, effective therapies. Extravasation, an essential rate-limiting process in the metastatic cascade, includes three tightly coordinated steps: cancer cell adhesion to the endothelium, trans-endothelial migration, and early invasion into the secondary site. Focal adhesion proteins, including Tln1 and FAK, regulate the cytoskeleton dynamics: dysregulation of these proteins is often associated with metastatic progression and poor prognosis.Here, we studied the previously unexplored role of these targets in each extravasation step using engineered 3D in vitro models, which recapitulate the physiological vascular niche experienced by cancer cells during hematogenous metastasis.Human breast cancer and fibrosarcoma cell lines respond to Cdk5/Tln1/FAK axis perturbation, impairing their metastatic potential. Vascular breaching requires actin polymerization-dependent invadopodia formation. Invadopodia generation requires the structural function of FAK and Tln1 rather than their activation through phosphorylation. Our data support that the inhibition of FAKS732 phosphorylation delocalizes ERK from the nucleus, decreasing ERK phosphorylated form. These findings indicate the critical role of these proteins in driving trans-endothelial migration. In fact, both knock-down experiments and chemical inhibition of FAK dramatically reduces lung colonization in vivo and TEM in microfluidic setting. Altogether, these data indicate that engineered 3D in vitro models coupled to in vivo models, genetic, biochemical, and imaging tools represent a powerful weapon to increase our understanding of metastatic progression.These findings point to the need for further analyses of previously overlooked phosphorylation sites of FAK, such as the serine 732, and foster the development of new effective antimetastatic treatments targeting late events of the metastatic cascade.

Cancer research has always relied on the use of in vitro models that enable the study of cells outside of the human body to discover drugs, improve cellular therapy and diagnosis. Now that it is established that the cellular and biochemical microenvironment in the body can influence cancer development, in vitro models must account for such factors. It follows in vitro models have become more complex with time, evolving from 2D models toward more physiologically relevant 3D assays. Here, we review 2D assays that have and are still helping scientists unravel mechanisms underlying cancer. We expose the reasons for switching to this more sophisticated art of cell culture. We provide details about the different formats of 3D cell culture for cancer research, ranging from spheroids, to scaffolds and microfluidic models. We illustrate how such assays have been used to dissect the entire process of cancer development, with particular emphasis on the metastatic cascade. Finally, we expose the latest trends in 3D cell culture that promise to become essential to the study of cancer and to the onset of personalized medicine, such as the development of stem-cell-derived organoids.

The ability of a cancer cell to migrate through the dense extracellular matrix within and surrounding the solid tumor is a critical determinant of metastasis. Macrophages enhance invasion and metastasis in the tumor microenvironment, but the basis for their effects is not fully understood. Using a microfluidic 3D cell migration assay, we found that the presence of macrophages enhanced the speed and persistence of cancer cell migration through a 3D extracellular matrix in a matrix metalloproteinases (MMP)-dependent fashion. Mechanistic investigations revealed that macrophage-released TNFα and TGFβ1 mediated the observed behaviors by two distinct pathways. These factors synergistically enhanced migration persistence through a synergistic induction of NF-κB-dependent MMP1 expression in cancer cells. In contrast, macrophage-released TGFβ1 enhanced migration speed primarily by inducing MT1-MMP expression. Taken together, our results reveal new insights into how macrophages enhance cancer cell metastasis, and they identify TNFα and TGFβ1 dual blockade as an antimetastatic strategy in solid tumors., National Institutes of Health (U.S.) (Grant U01 CA202177-01)

Metastasis is the leading cause of cancer-related deaths. Recent developments in cancer immunotherapy have shown exciting therapeutic promise for metastatic patients. While most therapies target T cells, other immune cells, such as monocytes, hold great promise for therapeutic intervention. In our study, we provide primary evidence of direct engagement between human monocytes and tumor cells in a 3D vascularized microfluidic model. We first characterize the novel application of our model to investigate and visualize at high resolution the evolution of monocytes as they migrate from the intravascular to the extravascular micro-environment. We also demonstrate their differentiation into macrophages in our all-human model. Our model replicates physiological differences between different monocyte subsets. In particular, we report that inflammatory, but not patrolling, monocytes rely on actomyosin based motility. Finally, we exploit this platform to study the effect of monocytes, at different stages of their life cycle, on cancer cell extravasation. Our data demonstrates that monocytes can directly reduce cancer cell extravasation in a non-contact dependent manner. In contrast, we see little effect of monocytes on cancer cell extravasation once monocytes transmigrate through the vasculature and are macrophage-like. Taken together, our study brings novel insight into the role of monocytes in cancer cell extravasation, which is an important step in the metastatic cascade. These findings establish our microfluidic platform as a powerful tool to investigate the characteristics and function of monocytes and monocyte-derived macrophages in normal and diseased states. We propose that monocyte-cancer cell interactions could be targeted to potentiate the anti-metastatic effect we observe in vitro, possibly expanding the milieu of immunotherapies available to tame metastasis.

In recognition of the enormous potential of immunotherapies against cancer, research into the interactions between tumor and immune cells has accelerated, leading to the recent FDA approval of several drugs that reduce cancer progression. Numerous cellular and molecular interactions have been identified by which immune cells can intervene in the metastatic cascade, leading to the development of several in vivo and in vitro model systems that can recapitulate these processes. Among these, microfluidic technologies hold many advantages in terms of their unique ability to capture the essential features of multiple cell type interactions in three-dimensions while allowing tight control of the microenvironment and real-time monitoring. Here, we review current assays and discuss the development of new microfluidic technologies for immunotherapy. Keywords microfluidics immunotherapy metastatic cancer drug screening

The intracellular environment is composed of a filamentous network that exhibits dynamic turnover of cytoskeletal components and internal force generation from molecular motors. Particle tracking microrheology enables a means to probe the internal mechanics and dynamics. Here, we develop an analytical model to capture the basic features of the active intracellular mechanical environment, including both thermal and motor-driven effects, and show consistency with a diverse range of experimental microrheology data. We further perform microrheology experiments, integrated with Brownian dynamics simulations of the active cytoskeleton, on metastatic breast cancer cells embedded in a three-dimensional collagen matrix with and without the presence of epidermal growth factor to probe the intracellular mechanical response in a physiologically mimicking scenario. Our results demonstrate that EGF stimulation can alter intracellular stiffness and power output from molecular motor-driven fluctuations in cells overexpressing an invasive isoform of the actin-associated protein Mena.

Mice are a common animal model for studies of aqueous humor dynamics and outflow physiology. The anatomy of the conventional outflow pathway in mice is similar to that of humans with a continuous Schlemm's canal and lamellated trabecular meshwork.1 Like primates, mice possess a ciliary muscle that forms tendinous connections to the elastic fiber net of the trabecular meshwork and the inner wall endothelium of Schlemm's canal.2 Compounds that affect outflow facility in humans, including pilocarpine,2,3 TGF-β2,4,5 latanoprost6–8 prostaglandin EP4 receptor agonist,9–11 and sphingosine 1-phosphate,11,12 similarly affect outflow facility in mice. Recently, mice have been used to validate novel compounds that increase outflow facility based on hits from screening assays of cellular contractility.13 Numerous investigators have measured outflow facility in mice.2,5,6,8,11,14–26 However, on account of the small dimensions of the mouse eye and the low flow rates involved, there is greater potential for physical factors to influence ocular perfusion measurements in mice compared with larger species. For example, evaporation from the surface of the eye, which is more pronounced in smaller eyes that have a larger surface to volume ratio, may lead to dehydration of the corneoscleral shell and artifactually increase the apparent outflow rate. Importantly, this effect would manifest as a pressure-independent outflow during perfusion. Posterior bowing of the iris, known as anterior chamber (AC) deepening, artificially increases outflow facility by applying traction to the trabecular meshwork.27,28 Anterior chamber deepening typically occurs during ocular perfusion via the AC, when the pressure in the AC exceeds that in the posterior chamber (PC). The pressure difference causes the iris–lens channel to collapse like a 1-way valve, preventing pressure equilibration across the iris. Anterior chamber deepening can be prevented by perfusion via the PC, by creating a fluidic shunt across the iris,29 or by iridectomy.30 Note that AC deepening is not synonymous with ‘AC depth,' which represents the distance between the posterior cornea and anterior lens. Because mice have a relatively large crystalline lens, it has been proposed that AC deepening may be negligible in mice,18 but this has not been specifically examined. To address these gaps in knowledge, this study examined the influence of hydration and AC deepening on pressure-dependent and pressure-independent outflow in enucleated mouse eyes. We also examined the effect of temperature that could account for up to 40% variation in apparent outflow facility due to changes in water viscosity between room and physiological temperature.

Intravital imaging can be performed in mice and has proven extremely useful in uncovering the role of immune cells in cancer metastasis, but requires specialized expertise and equipment. In the last decade, microfluidic models have been developed that replicate key aspects of the metastastic microenvironment, allowing for higher resolution imaging than in vivo studies, and for enhanced control over the biochemical and physical microenvironment of the assay at cellular length scales in 3D. This is particularly useful to studying metastasis, which is composed of several hidden steps such as intravasation or extravasation that can be rare and thus difficult to image in vivo; in contrast, this rate of occurrence can be more readily modulated in vitro. In this study, we use a microfluidic model to investigate the role of monocytes in cancer cell extravasation. In mouse studies, monocytes have recently been shown to have either pro- or anti-metastatic functions depending on their phenotype; however, the underlying mechanisms remain unclear, and in particular, whether the monocytes helped during extravasation or at later stages. We propose to investigate this in our microfluidic models, so as to find ways to therapeutically block immune-mediated extravasation as a means to reduce metastasis. A previously developed microfluidic assay was used to form a 3D microvascular network (MVN) that consists of 3 separate compartments, flanked by media channels connected to reservoirs. In the two lateral compartments, normal human lung fibroblasts were seeded at 5 million/ml, while in the central compartment GFP HUVECs were placed at 4 million/ml all in a 3D fibrin gel. After 4 days, the HUVECs self-assemble into a MVN, which forms lumens that open directly to the media channel. Therefore, cancer cells can be perfused within the MVN via the reservoirs, and their transendothelial migration is tracked by confocal imaging over time for up to 12 hours. Monocytes were perfused with cancer cells in the MVN to test whether the monocytes could affect cancer cell extravasation intra-luminally, as they would in the blood circulation. In addition, we sort the monocytes into classical or inflammatory monocytes and perfuse them separately with MDA-MB-231. Both monocytes and macrophages were derived from healthy donors blood. Monocytes were found to significantly decrease cancer cell extravasation. 11 hrs after cancer cells were perfused with monocytes inside the MVN, 19.8±6.1 % of MDA-MB-231 had extravasated vs. 53.5±11% when cancer cells were perfused alone (p=0.041, unpaired t-test). When monocytes were perfused alone, most of them had extravasated 4 days later (94±2%). Interestingly, 46.4±3.1 % stained positive for MRC-1, suggesting the monocytes had differentiated into M2-like macrophages following extravasation. In addition, MRC-1 positive macrophages were significantly closer to the vascular network than macrophages that did not express MRC-1 (37.1±7.1 vs. 65.9±9.7 µm, p =0.02, unpaired t-test). One day after perfusion, only 33% of classical monocytes had extravasated vs. 77% of inflammatory monocytes. Inflammatory monocytes migrated much faster (1.12±0.85 vs 0.33±0.20 µm.min, respectively, p =0.0005, unpaired t-test). Current work will reveal whether each subset has different effects on cancer cell extravasation. Citation Format: Alexandra Boussommier-Calleja, Roger Kamm. Role of monocytes in 3D microfluidic models of cancer cell extravasation. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B22.

To determine whether dexamethasone (DEX)-induced ocular hypertension (OHT) in mice mimics the hallmarks of steroid-induced glaucoma (SIG) in humans, including reduced conventional outflow facility (C), increased extracellular matrix (ECM), and myofibroblasts within the outflow pathway.Osmotic mini-pumps were implanted subcutaneously into C57BL/6J mice for systemic delivery of DEX (3-4 mg/kg/d, n = 31 mice) or vehicle (n = 28). IOP was measured weekly by rebound tonometry. After 3 to 4 weeks, mice were euthanized and eyes enucleated for ex vivo perfusion to measure C, for electron microscopy to examine the trabecular meshwork (TM) and Schlemm's canal (SC), or for immunohistochemistry to examine type IV collagen and α-smooth muscle actin. The length of basement membrane material (BMM) was measured along the anterior-posterior extent of SC by electron microscopy. Ultrastructural changes in BMM of DEX-treated mice were compared against archived human SIG specimens.Dexamethasone increased IOP by 2.6 ± 1.6 mm Hg (mean ± SD) over 3 to 4 weeks and decreased C by 52% ± 17% versus controls. Intraocular pressure elevation correlated with decreased C. Dexamethasone treatment led to increased fibrillar material in the TM, plaque-like sheath material surrounding elastic fibers, and myofibroblasts along SC outer wall. The length of BMM underlying SC was significantly increased in mice with DEX and in humans with SIG, and in mice decreased C correlated with increased BMM.Dexamethasone-induced OHT in mice mimics hallmarks of human SIG within 4 weeks of DEX treatment. The correlation between reduced C and newly formed ECM motivates further study using DEX-treated mice to investigate the pathogenesis of conventional outflow obstruction in glaucoma.

Recent in vivo mouse studies have revealed that macrophages can promote metastasis, while certain subsets of monocytes either help or block metastasis. Further studies are warranted to understand the underlying mechanisms, so as to target pharmacologically these immune cells as a means to halt metastasis. Intravital imaging can be performed in mice and has proven extremely useful in uncovering the role of macrophage and monocytes in metastasis, but requires specialized expertise and equipment. Concurrently, microfluidic models have been developed to dissect certain steps of the metastatic spread, because they allow for easier imaging at high resolution and facilitate an improved microenvironment control over more traditional macro-scale in vitro systems. In this study, a microfluidic model was used to investigate the effects of monocytes and macrophages on cancer cell extravasation. A previously developed microfluidic assay was used to form a 3D microvascular network (MVN) that consists of 3 separate compartments, flanked by media channels connected to reservoirs. In the two lateral compartments, normal human lung fibroblasts were seeded at 5 million/ml, while in the central compartment GFP HUVECs were placed at 4 million/ml all in a 3D fibrin gel. After 4 days, the HUVECs self-assemble into a MVN, which forms lumens that open directly to the media channel. Therefore, cancer cells can be perfused within the MVN via the reservoirs, and their transendothelial migration is tracked by confocal imaging over time for up to 12 hours. In this study, we mix the HUVECs with the macrophages (ratio 40:1) such that the macrophages are found in the fibrin on the outside of the vessels. Alternatively, we perfuse monocytes in the MVN, most of which have extravasated 4 days later (94±2%), thus replicating macrophage differentiation from recruited monocytes. In another study, monocytes were perfused with cancer cells in the MVN to test whether the monocytes could affect cancer cell extravasation intra-luminally, as they would in the blood circulation. Both monocytes and macrophages were derived from healthy donors' blood. Monocyte-derived macrophages (MDMs) were on average 33.4±4.4 μm (mean±SEM) away from the MVN, of which 40% were found within 10 μm. Interestingly, 46.4±3.1% stained positive for MRC-1. The MDM:MDA-MB-231 ratio was 2±0.5. The presence of MDM around the microvascular networks did not affect cancer cell extravasation rate; 10 hours after the cancer cells were perfused within the MVN, 57±15 vs. 60±6% of MDA-MB-231 had extravasated with or without MDMs, respectively. 11±6% of MDA-MB-231s came in contact with MDMs, for 5.1±0.6 hrs. Contact was mostly established after cancer cells had extravasated (73±19% of cancer cells), and less often while the cancer cell was extravasating (15±9%) or inside the vessels (12±12%). At t = 0, the average shortest distance between a MDM and a cancer cell arrested within the vessels was 98.4±6.3 μm, and was the same for cancer cells that did or did not eventually extravasate. However, the cancer cells that extravasated migrated further away from the μVN surface in presence of MDMs; 4.5 hrs after extravasation, cells were found 16.5±4.9 vs. 5.4±2.1 μm away from the endothelial wall with or without MDMs, respectively (p = 0.044, unpaired student t-test). In a separate study, monocytes were found to significantly decrease cancer cell extravasation. 11 hrs after cancer cells were perfused with monocytes inside the MVN, 19.8±6.1% of MDA-MB-231 had extravasated vs. 53.5±11% when cancer cells were perfused alone (p = 0.041, unpaired t-test). In conclusion, monocytes but not macrophage seem to affect cancer cell extravasation. Further studies will generalize the results to other cancer cell lines, and investigate how monocytes might decrease extravasation, and how this effect could be exacerbated pharmacologically to decrease metastasis. Citation Format: Alexandra Boussommier-Calleja, Roger D. Kamm. The role of macrophages and monocytes during cancer cell extravasation in 3D vascularized microfluidic models [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A126.

Purpose Mouse models are useful for glaucoma research, but it is unclear whether intraocular pressure (IOP) regulation in mice operates through mechanisms similar to those in humans. Our goal was to determine whether pharmacologic compounds that affect conventional outflow facility in human eyes exert similar effects in C57BL/6 mice. Methods A computerized perfusion system was used to measure conventional outflow facility in enucleated mouse eyes ex vivo. Paired eyes were perfused sequentially, either immediately after enucleation or after 3 hours storage at 4°C. Three groups of experiments examined sphingosine 1-phosphate (S1P), S1P with antagonists to S1P(1) and S1P(2) receptors, and the prostanoid EP(4) receptor agonist 3,7-dithia PGE(1). We also examined whether a 24-hour postmortem delay affected the response to 3,7-dithia prostaglandin E(1) (PGE(1)). Results S1P decreased facility by 39%, and was blocked almost completely by an S1P(2), but not S1P(1), receptor antagonist. The S1P(2) receptor antagonist alone increased facility nearly 2-fold. 3,7-dithia PGE(1) increased facility by 106% within 3 hours postmortem. By 24 hours postmortem, the facility increase caused by 3,7-dithia PGE(1) was reduced 3-fold, yet remained statistically detectable. Conclusions C57BL/6 mice showed opposing effects of S1P(2) and EP(4) receptor activation on conventional outflow facility, as observed in human eyes. Pharmacologic effects on facility were detectable up to 24 hours postmortem in enucleated mouse eyes. Mice are suitable models to examine the pharmacology of S1P and EP(4) receptor stimulation on IOP regulation as occurs within the conventional outflow pathway of human eyes, and are promising for studying other aspects of aqueous outflow dynamics.

Osteoarthritis (OA) is a degenerative joint disease that is a leading cause of adult pain and disability in Western countries1. Clinically, several structural features of the joint are important in diagnosis, prognosis and evaluation of treatment efficacy, e.g. cartilage volume, homogeneity and joint space narrowing2,3. In animal models of OA, structural features such as bone defects and cartilage changes are commonly investigated using histomorphometry, a technique that uses stereological point counting and manual tracing of regions of interest to extract 3-dimensional (3D) geometrical properties from 2D histology slides. This is time-consuming and subject to inter-observer variations, hence limiting precision 4,5. Therefore, a technique for rapidly imaging joint structures in 3D at high resolution, including articular cartilage and subchondral bone, is currently needed in pre-clinical OA research.Copyright © 2011 by ASME

Mice are commonly used in glaucoma research, but relatively little is known about aqueous outflow dynamics in the species. To facilitate future use of the mouse as a model of aqueous humor outflow, several fundamental physiological parameters were measured in the mouse eye.Eyes from adult mice of either sex (C57BL/6 background) were enucleated, cannulated with a 33-gauge needle, and perfused at constant pressure while inflow was continuously measured.At 8 mm Hg, total outflow facility (C(total)) was 0.022 ± 0.005 μL/min/mm Hg (all values mean ± SD; n = 21). The flow-pressure relationship was linear up to 35 mm Hg. The conventional outflow facility (C(conv)) was 0.0066 ± 0.0009 μL/min/mm Hg, and the unconventional outflow (F(u)) was 0.114 ± 0.019 μL/min, both measured at room temperature. At 8 mm Hg, 66% of the outflow was via the unconventional pathway. In a more than 2-hour-long perfusion at 8 mm Hg, the rate of facility change was 2.4% ± 5.4% (n = 11) of starting facility per hour. The ocular compliance (0.086 ± 0.017 μL/mm Hg; n = 5) was comparable to the compliance of the perfusion system (0.100 ± 0.004 μL/mm Hg).Mouse eyes are similar to human eyes, in that they have no detectable washout rate and a linear pressure-flow relationship over a broad range of intraocular pressures. Because of the absence of washout and the apparent presence of a true Schlemm's canal, the mouse is a useful model for studying the physiology of the inner wall of Schlemm's canal and the conventional outflow tissues.

PURPOSE Intraocular pressure (IOP) varies between genetically distinct strains of mice. The purpose was to test the hypothesis that strain-dependent differences in IOP are attributable to differences in conventional outflow facility (C). METHODS The IOP was measured by rebound tonometry in conscious or anesthetized BALB/cJ, C57BL/6J, and CBA/J mice (N = 6-10 per strain). Conventional outflow facility was measured by ex vivo perfusion of enucleated eyes (N = 9-10 per strain). RESULTS Conscious IOP varied between strains, being highest in CBA/J (14.5 ± 0.9 mm Hg, mean ± SD), intermediate in C57BL/6J (12.3 ± 1.0 mm Hg), and lowest in BALB/cJ (10.6 ± 1.8 mm Hg) mice. Anesthesia reduced IOP and eliminated any detectable differences between strains. Conventional outflow facility also varied between strains, but, in contrast to IOP, C was lowest in CBA/J (0.0113 ± 0.0031 μL/min/mm Hg) and highest in BALB/cJ (0.0164 ± 0.0059 μL/min/mm Hg). Like IOP, C was intermediate in C57BL/6J (0.0147 ± 0.0029 μL/min/mm Hg). There was a strong correlation between conscious IOP and outflow resistance (1/C) from individual eyes across all three strains, revealing that 70% of the variation in IOP was attributable to variation in outflow resistance. CONCLUSIONS Differences in IOP among three genetically distinct murine strains are attributable largely to differences in conventional outflow facility. These results motivate further studies using mice to identify the morphologic and genetic factors that underlie IOP regulation within the conventional outflow pathway.