Neutrophil elastase as a versatile cleavage enzyme for activation of αvß3 integrin-targeted small molecule drug conjugates with different payload classes in the tumor microenvironment.

Introduction: The development of bioconjugates for the targeted delivery of anticancer agents is gaining momentum after recent success of antibody drug conjugates (ADCs) in the clinic. Smaller format conjugates may have several advantages including better tumor penetration; however, cellular uptake and trafficking may be substantially different from ADCs. To fully leverage the potential of small molecule drug conjugates (SMDCs) with potent binding molecules mediating tumor homing, novel linker chemistries susceptible for efficient extracellular activation and payload release in the tumor microenvironment (TME) need to be explored. Methods: We designed a novel class of SMDCs, which target αvß3 integrins for tumor homing and are cleaved by neutrophil elastase (NE), a serine protease active in the TME. A peptidomimetic αvß3 ligand was attached via optimized linkers composed of substrate peptide sequences of NE connected to different functional groups of various payload classes, such as camptothecins, monomethyl auristatin E, kinesin spindle protein inhibitors (KSPi) and cyclin-dependent kinase 9 inhibitors (CDK-9i). Results: NE-mediated cleavage was found compatible with the diverse linker attachments via hindered ester bonds, amide bonds and sulfoximide bonds. Efficient and traceless release of the respective payloads was demonstrated in biochemical assays. The newly designed SMDCs were highly stable in buffer as well as in rat and human plasma. Cytotoxicity of the SMDCs in cancer cell lines was clearly dependent on NE. IC50 values were in the nanomolar or sub-nanomolar range across several cancer cell lines reaching similar potencies as compared to the respective payloads only in the presence of NE. In vivo pharmacokinetics evaluating SMDC and free payload exposures in rat and particularly the robust efficacy with good tolerability in triple negative breast and small cell lung cancer murine models demonstrate the utility of this approach for selective delivery of payloads to the tumor. Discussion: These results highlight the broad scope of potential payloads and suitable conjugation chemistries paving the way for future SMDCs harnessing the safety features of targeted delivery approaches in combination with NE cleavage in the TME.

Cusatuzumab plus azacitidine in newly diagnosed acute myeloid leukaemia ineligible for intensive chemotherapy (CULMINATE): part one of a randomised, phase 2, dose optimisation study.

BACKGROUND: Cusatuzumab, a high-affinity anti-CD70 antibody, has shown preliminary activity as a treatment for acute myeloid leukaemia when combined with azacitidine. We aimed to determine the optimum dose for future trials of cusatuzumab in combination with azacitidine in patients with previously untreated acute myeloid leukaemia who are not eligible for intensive chemotherapy. METHODS: In this randomised, phase 2, open-label, dose-optimisation study we enrolled adult patients aged 18 years or older with newly diagnosed acute myeloid leukaemia not eligible for intensive chemotherapy, and with Eastern Cooperative Oncology Group scores of 0-2, from 40 hospitals and centres across seven countries. In part one of the trial, participants were randomly allocated 1:1 to 10 mg/kg or 20 mg/kg intravenous cusatuzumab on days 3 and 17, combined with subcutaneous or intravenous azacitidine 75 mg/m2 on days 1-7 in 28-day cycles. The primary efficacy outcome was the rate of complete remission in the intention-to-treat group. The two dose cohorts were evaluated independently without between-cohort statistical comparison. Safety analyses were performed in all patients who received one dose of study drug. Part two of the trial was planned to be a single-arm expansion to evaluate cusatuzumab plus azacitidine at the cusatuzumab dose level selected in part one (primary hypothesis ≥35% rate of complete remission vs null hypothesis of 20%); however, changes in the acute myeloid leukaemia treatment landscape during this trial made it unlikely that enrolment to part two of the study would be clinically feasible, so the study stopped at the end of part one. The trial was registered at ClinicalTrials.gov, NCT04023526. FINDINGS: 103 patients were enrolled between Aug 30, 2019, and Feb 25, 2020, and randomly assigned to either cusatuzumab 10 mg/kg (n=51) or 20 mg/kg (n=52). Median follow-up was 7·2 months (IQR 10·7 months). 57 of 103 (55%) patients were male and 46 (45%) patients were female, 78 (76%) were White, one (1%) was Asian, and 24 (23%) did not report their race. In the 10 mg/kg group, complete remission rate was 12% (six of 51 patients; 95% CI 6-23) and in the 20 mg/kg group was 27% (14 of 52; 17-40). Grade 3 or worse treatment-emergent adverse events (TEAEs) were similar between the cusatuzumab 10 mg/kg (n=51) and 20 mg/kg (n=51) cohorts and included thrombocytopenia (24 patients [47%] vs 29 [57%]), anaemia (24 [47%] vs 17 [33%]), and neutropenia (20 [39%] in both cohorts). Serious TEAEs were also similar in the two cohorts (44 [86%] vs 40 [78%]). Treatment-related TEAEs leading to death were reported in both groups (three patients [6%] in the 10 mg/kg group vs one patient [2%] in the 20 mg/kg group); the reported causes of death were pneumonia (n=2) and septic shock (n=2). INTERPRETATION: Although part one of this study was not designed to formally compare the two dose cohorts for efficacy, the totality of clinical data for cusatuzumab studies performed to date indicate that cusatuzumab 20 mg/kg plus azacitidine represents the optimal dose for further studies. A phase 1b study investigating the triple combination of cusatuzumab with venetoclax and azacitidine is underway (NCT04150887). FUNDING: Janssen Research & Development and argenx.

Enitociclib, a Selective CDK9 Inhibitor, Induces Complete Regression of MYC+ Lymphoma by Downregulation of RNA Polymerase II Mediated Transcription.

Double-hit diffuse large B-cell lymphoma (DH-DLBCL) is an aggressive, and often refractory, type of B-cell non-Hodgkin lymphoma (NHL) characterized by rearrangements in MYC and BCL2. Cyclin-dependent kinase 9 (CDK9) regulates transcriptional elongation and activation of transcription factors, including MYC, making it a potential targeted approach for the treatment of MYC+ lymphomas. Enitociclib is a well-tolerated and clinically active CDK9 inhibitor leading to complete metabolic remissions in 2 of 7 patients with DH-DLBCL treated with once weekly 30 mg intravenous administration. Herein, we investigate the pharmacodynamic effect of CDK9 inhibition in preclinical models and in blood samples from patients [DH-DLBCL (n = 10) and MYC+ NHL (n = 5)] treated with 30 mg i.v. once weekly enitociclib. Enitociclib shows significant regulation of RNA polymerase II Ser2 phosphorylation in a MYC-amplified SU-DHL-4 cell line and depletion of MYC and antiapoptosis protein MCL1 in SU-DHL-4 and MYC-overexpressing SU-DHL-10 cell lines in vitro. Tumor growth inhibition reaching 0.5% of control treated SU-DHL-10 xenografts is achieved in vivo and MYC and MCL1 depletion as well as evidence of apoptosis activation after enitociclib treatment is demonstrated. An unbiased analysis of the genes affected by CDK9 inhibition in both cell lines demonstrates that RNA polymerase II and transcription pathways are primarily affected and novel enitociclib targets such as PHF23 and TP53RK are discovered. These findings are recapitulated in blood samples from enitociclib-treated patients; while MYC downregulation is most robust with enitociclib treatment, other CDK9-regulated targets may be MYC independent delivering a transcriptional downregulation via RNA polymerase II. SIGNIFICANCE: MYC+ lymphomas are refractory to standard of care and novel treatments that downregulate MYC are needed. The utility of enitociclib, a selective CDK9 inhibitor in this patient population, is demonstrated in preclinical models and patients. Enitociclib inhibits RNA polymerase II function conferring a transcriptional shift and depletion of MYC and MCL1. Enitociclib intermittent dosing downregulates transcription factors including MYC, providing a therapeutic window for durable responses in patients with MYC+ lymphoma.

Chronic exposure of mice to phthalates enhances TGF beta signaling and promotes uterine fibrosis.

Phthalates are synthetic chemicals widely used as plasticizers and stabilizers in various consumer products. Because of the extensive production and use of phthalates, humans are exposed to these chemicals daily. While most studies focus on a single phthalate, humans are exposed to a mixture of phthalates on a regular basis. The impact of continuous exposure to phthalate mixture on uterus is largely unknown. Thus, we conducted studies in which adult female mice were exposed for 6 months to 0.15 ppm and 1.5 ppm of a mixture of phthalates via chow ad libitum. Our studies revealed that consumption of phthalate mixture at 0.15 ppm and 1.5 ppm for 6 months led to a significant increase in the thickness of the myometrial layer compared to control. Further investigation employing RNA-sequencing revealed an elevated transforming growth factor beta (TGF-ß) signaling in the uteri of mice fed with phthalate mixture. TGF-ß signaling is associated with the development of fibrosis, a consequence of excessive accumulation of extracellular matrix components, such as collagen fibers in a tissue. Consistent with this observation, we found a higher incidence of collagen deposition in uteri of mice exposed to phthalate mixture compared to unexposed controls. Second Harmonic Generation (SHG) imaging showed disorganized collagen fibers and nanoindentation indicated a local increase in uterine stiffness upon exposure to phthalate mixture. Collectively, our results demonstrate that chronic exposure to phthalate mixture can have adverse effects on uterine homeostasis.

Discovery of VIP236, an αvß3-Targeted Small-Molecule-Drug Conjugate with Neutrophil Elastase-Mediated Activation of 7-Ethyl Camptothecin Payload for Treatment of Solid Tumors.

The emerging field of small-molecule-drug conjugates (SMDCs) using small-molecule biomarker-targeted compounds for tumor homing may provide new perspectives for targeted delivery. Here, for the first time, we disclose the structure and the synthesis of VIP236, an SMDC designed for the treatment of metastatic solid tumors by targeting αvß3 integrins and extracellular cleavage of the 7-ethyl camptothecin payload by neutrophil elastase in the tumor microenvironment. Imaging studies in the Lewis lung mouse model using an elastase cleavable quenched substrate showed pronounced elastase activity in the tumor. Pharmacokinetics studies of VIP236 in tumor-bearing mice demonstrated high stability of the SMDC in plasma and high tumor accumulation of the cleaved payload. Studies in bile-duct-cannulated rats showed that biliary excretion of the unmodified conjugate is the primary route of elimination. Treatment- and time-dependent phosphorylation of H2AX, a marker of DNA damage downstream of topoisomerase 1 inhibition, verified the on-target activity of the payload cleaved from VIP236 in vivo. Treatment with VIP236 resulted in long-lasting tumor regression in subcutaneous patient-derived xenograft (PDX) models from patients with non-small-cell lung, colon, and renal cancer as well as in two orthotopic metastatic triple-negative breast cancer PDX models. In these models, a significant reduction of brain and lung metastases also was observed.

Improving visualization of the cervix during pelvic exams: A simulation using a physical model of the speculum and human vagina as a steppingstone to reducing disparities in gynecological cancers.

Pelvic exams are frequently complicated by collapse of the lateral vaginal walls, obstructing the view of the cervix. To overcome this, physicians frequently repurpose a glove or a condom as a sheath placed over the speculum blades to retract the lateral vaginal walls. Despite their regular use in clinical practice, little research has been done comparing the relative efficacy of these methods. Better visualization of the cervix can benefit patients by decreasing examination-related discomfort, improving cancer screening accuracy, and preventing the need to move the examination to the operating room under general anesthesia. This study presents a physical model that simulates vaginal pressure being exerted around a speculum. Using it, we conduct controlled experiments comparing the efficacy of different condom types, glove materials, glove sizes, and techniques to place gloves on the speculum. The results show that the best sheath is the middle finger of nitrile-material gloves. They provide adequate lateral wall retraction without significantly restricting the opening of the speculum. In comparison, condoms provide a smaller amount of retraction due to loosely fitting the speculum. They may still be a reasonable option for a different speculum size. However, vinyl-material gloves are an impractical option for sheaths; they greatly restrict speculum opening, occasionally even breaking the speculum, which overcome its retraction benefits. Glove size, condom brand, and condom material (latex vs polyisoprene) had minimal impact. This study serves as a guide for clinicians as they use easily accessible tools to perform difficult pelvic exams. We recommend that physicians consider nitrile gloves as the preferred option for a sheath around a speculum. Additionally, this study demonstrates proof-of-concept of a physical model that quantitatively describes different materials on their ability to improve cervical access. This model can be used in future research with more speculum and material combinations, including with materials custom-designed for vaginal retraction.

VIP152 is a selective CDK9 inhibitor with pre-clinical in vitro and in vivo efficacy in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is effectively treated with targeted therapies including Bruton tyrosine kinase inhibitors and BCL2 antagonists. When these become ineffective, treatment options are limited. Positive transcription elongation factor complex (P-TEFb), a heterodimeric protein complex composed of cyclin dependent kinase 9 (CDK9) and cyclin T1, functions to regulate short half-life transcripts by phosphorylation of RNA Polymerase II (POLII). These transcripts are frequently dysregulated in hematologic malignancies; however, therapies targeting inhibition of P-TEFb have not yet achieved approval for cancer treatment. VIP152 kinome profiling revealed CDK9 as the main enzyme inhibited at 100 nM, with over a 10-fold increase in potency compared with other inhibitors currently in development for this target. VIP152 induced cell death in CLL cell lines and primary patient samples. Transcriptome analysis revealed inhibition of RNA degradation through the AU-Rich Element (ARE) dysregulation. Mechanistically, VIP152 inhibits the assembly of P-TEFb onto the transcription machinery and disturbs binding partners. Finally, immune competent mice engrafted with CLL-like cells of Eµ-MTCP1 over-expressing mice and treated with VIP152 demonstrated reduced disease burden and improvement in overall survival compared to vehicle-treated mice. These data suggest that VIP152 is a highly selective inhibitor of CDK9 that represents an attractive new therapy for CLL.

Millimeter-scale topography facilitates coral larval settlement in wave-driven oscillatory flow.

Larval settlement in wave-dominated, nearshore environments is the most critical life stage for a vast array of marine invertebrates, yet it is poorly understood and virtually impossible to observe in situ. Using a custom-built flume tank that mimics the oscillatory fluid flow over a shallow coral reef, we isolated the effect of millimeter-scale benthic topography and showed that it increases the settlement of slow-swimming coral larvae by an order of magnitude relative to flat substrates. Particle tracking velocimetry of flow fields revealed that millimeter-scale ridges introduced regions of flow recirculation that redirected larvae toward the substrate surface and decreased the local fluid speed, effectively increasing the window of time for larvae to settle. Regions of recirculation were quantified using the Q-criterion method of vortex identification and correlated with the settlement locations of larvae for the first time. In agreement with experiments, computational fluid dynamics modeling and agent-based larval simulations also showed significantly higher settlement onto ridged substrates. Additionally, in contrast to previous reports on the effect of micro-scale substrate topography, we found that these topographies did not produce key hydrodynamic features linked to increased settlement. These findings highlight how physics-based substrate design can create new opportunities to increase larval recruitment for ecosystem restoration.

DNA Origami Nanostructures Elicit Dose-Dependent Immunogenicity and Are Nontoxic up to High Doses In Vivo.

DNA origami (DO) nanotechnology enables the construction of precise nanostructures capable of functionalization with small molecule drugs, nucleic acids, and proteins, suggesting a promising platform for biomedical applications. Despite the potential for drug and vaccine delivery, the impact of DO vehicles on immunogenicity in vivo is not well understood. Here, two DO vehicles, a flat triangle and a nanorod, at varying concentrations are evaluated in vitro and with a repeated dosing regimen administered at a high dose in vivo to study early and late immunogenicity. The studies show normal CD11b+ myeloid cell populations preferentially internalize DO in vitro. DO structures distribute well systemically in vivo, elicit a modest pro-inflammatory immune response that diminishes over time and are nontoxic as shown by weight, histopathology, lack of cytokine storm, and a complete biochemistry panel at the day 10 end point. The results take critical steps to characterize the biological response to DO and suggest that DO vehicles represent a promising platform for drug delivery and vaccine development where immunogenicity should be a key consideration.

Simulated confluence on micropatterned substrates correlates responses regulating cellular differentiation.

While cells are known to behave differently based on the size of micropatterned islands, and this behavior is thought to be related to cell size and cell-cell contacts, the exact threshold for this difference between small and large islands is unknown. Furthermore, while cell size and cell-cell contacts can be easily manipulated on small islands, they are harder to measure and continually monitor on larger islands. To investigate this size threshold, and to explore cell size, cell-cell contacts, and differentiation, we use a previously established simulation to plan experiments and explain results that we could not explain from experiments alone. We use five seeding densities covering three orders of magnitude over 25-500 µm diameter islands to examine markers of proliferation and differentiation in bone marrow-derived mesenchymal cells (cell line). We show that osteogenic markers are most accurately described as a function of confluence for larger islands, but a function of time for smaller islands. We further show, using results of the simulation, that cell size and cell-cell contacts are also related to confluence on larger islands, but only cell-cell contacts are related to confluence on small islands. This study uses simulations to explain experimental results that could not be explained from experiments alone. Together, the simulations and experiments in this study show different differentiation patterns on large and small islands, and this simulation may be useful in planning future studies related to this study.

Morphological switch is associated with increase in cell-cell contacts, ALP, and confluence above a minimum island area to perimeter ratio.

During osteogenic differentiation in vitro, stem-like cells seeded at a low-density spread and are isolated. As the cells proliferate and mature, they become more cuboidal in shape with more cell-cell contacts. However, the coordination of this switch in cell morphology from elongated to cuboidal, cell-cell contacts, and differentiation is not known. In this work, we present results from experiments and a simulation of cell proliferation on protein-micropatterned islands that, independent of island size (25-1,000 µm) or shape (circles, squares, and hollow squares), shows a distinct morphological switch that is better described as a function of island confluence than time in culture, the standard measure in cell culture experiments. The simulation and experiments show cell morphology and island cell density versus confluence collapse to a single curve for all islands if the island area to perimeter ratio is ≥25 µm. Cell-cell contacts in the simulation and alkaline phosphatase (ALP) expression in experiments, a common marker for osteogenic differentiation, show exponential growth with confluence, rapidly increasing after the switch at ≈0.5 confluence. Furthermore, cell morphology, density, contacts, and ALP are better predicted by confluence than time in culture. The variability with time in culture leads to challenges in not only interpreting data but also in comparing data across research laboratories. This simulation can be used to predict cell behavior on different size and shape islands and to plan and optimize experiments that explore cell behavior as a function of a wide range of island geometries.

Interplay between integrins and cadherins to control bone differentiation upon BMP-2 stimulation.

Introduction: Upon BMP-2 stimulation, the osteoblastic lineage commitment in C2C12 myoblasts is associated with a microenvironmental change that occurs over several days. How does BMP-2 operate a switch in adhesive machinery to adapt to the new microenvironment and to drive bone cell fate is not well understood. Here, we addressed this question for BMP-2 delivered either in solution or physically bound of a biomimetic film, to mimic its presentation to cells via the extracellular matrix (ECM). Methods: Biommetics films were prepared using a recently developed automated method that enable high content studies of cellular processes. Comparative gene expressions were done using RNA sequencing from the encyclopedia of the regulatory elements (ENCODE). Gene expressions of transcription factors, beta chain (1, 3, 5) integrins and cadherins (M, N, and Cad11) were studied using quantitative PCR. ECM proteins and adhesion receptor expressions were also quantified by Western blots and dot blots. Their spatial organization in and around cells was studied using immuno-stainings. The individual effect of each receptor on osteogenic transcription factors and alkaline phosphatase expression were studied using silencing RNA of each integrin and cadherin receptor. The organization of fibronectin was studied using immuno-staining and quantitative microscopic analysis. Results: Our findings highlight a switch of integrin and cadherin expression during muscle to bone transdifferentiation upon BMP-2 stimulation. This switch occurs no matter the presentation mode, for BMP-2 presented in solution or via the biomimetic film. While C2C12 muscle cells express M-cadherin and Laminin-specific integrins, the BMP-2-induced transdifferentiation into bone cells is associated with an increase in the expression of cadherin-11 and collagen-specific integrins. Biomimetic films presenting matrix-bound BMP-2 enable the revelation of specific roles of the adhesive receptors depending on the transcription factor. Discussion: While ß3 integrin and cadherin-11 work in concert to control early pSMAD1,5,9 signaling, ß1 integrin and Cadherin-11 control RunX2, ALP activity and fibronectin organization around the cells. In contrast, while ß1 integrin is also important for osterix transcriptional activity, Cadherin-11 and ß5 integrin act as negative osterix regulators. In addition, ß5 integrin negatively regulates RunX2. Our results show that biomimetic films can be used to delinate the specific events associated with BMP-2-mediated muscle to bone transdifferentiation. Our study reveals how integrins and cadherins work together, while exerting distinct functions to drive osteogenic programming. Different sets of integrins and cadherins have complementary mechanical roles during the time window of this transdifferentiation.

Optimizing extracellular vesicles' isolation from chronic lymphocytic leukemia patient plasma and cell line supernatant.

In chronic lymphocytic leukemia (CLL) and very likely all cancer types, extracellular vesicles (EVs) are a common mechanism by which intercellular messages are communicated between normal, diseased, and transformed cells. Studies of EVs in CLL and other cancers have great variability and often lack reproducibility. For CLL patient plasma and cell lines, we sought to characterize current approaches used in isolating EV products and understand whether cell culture-conditioned media or complex biological fluids confound results. Utilizing nanoparticle tracking analysis, protein quantification, and electron microscopy, we show that ultracentrifugation with an OptiPrep cushion can effectively minimize contaminants from starting materials including plasma and conditioned media of CLL cell lines grown in EV-depleted complete RPMI media but not grown in the serum-free media AIM V commonly used in CLL experimental work. Moreover, we confirm the benefit of including 25 mM trehalose in PBS during EV isolation steps to reduce EV aggregation, to preserve function for downstream applications and characterization. Furthermore, we report the highest particles/µg EVs were obtained from our CLL cell lines utilizing the CELLine bioreactor flask. Finally, we optimized a proliferation assay that offers a functional evaluation of our EVs with minimal sample requirements.

Use of mean platelet volume in the assessment of intrauterine infection in newborns with combined thrombocytopenia and leukopenia at birth.

Objective: Intrauterine stress can be reflected on hematological disturbance at birth. Thrombocytopenia and leukopenia may be a result of exposure to maternal hypertensive disorders but may also indicate fetal inflammatory response to intrauterine infection, prompting empiric antibiotics use during the initial assessment after birth. Emerging data suggest long-term adverse health outcomes associated with antibiotics exposure early in life. In this report, we sought to assess the use of mean platelet volume (MPV) at birth in predicting fetal inflammatory response in newborns with combined thrombocytopenia and leukopenia.Materials and methods: This is a retrospective study from a single academic medical center. Data were collected prospectively on all newborns with thrombocytopenia and leukopenia within the first 24 h of life. The primary outcome was a composite of fetal tachycardia, premature preterm rupture of membrane with preterm labor, and histological evidence of chorioamnionitis/funisitis/villitis on placental pathology reports evaluated using a multiple logistical regression analysis.Results: The prevalence of combined thrombocytopenia and leukopenia was 5.8% (99 out of 1693 newborns) during the study period. The prevalence was highly associated with gestational age (R2 = 0.873). Twenty-four (32.4%) had abnormal MPV values at birth, defined as MPV > 9 or < 7 fL. Newborns with abnormal MPV had lower platelet counts and were more likely to have I:T ratio ≥0.2. In a univariate analysis, abnormal MPV (OR: 6.205, 95% CI: 1.923-20.022, p = .002), I:T ratio ≥0.2 (OR: 8.462, 95% CI: 1.396-51.281, p = .02), and platelet counts (OR: 98.4, 95% CI: 96.9%-99.9%, p = .035) were each significantly associated with a positive composite outcome. In a multivariate analysis, only abnormal MPV remained significantly associated with an increased likelihood of having a positive composite outcome, with an OR of 3.922 (95% CI: 1.094-14.06, p = .036).Conclusions: MPV may be a more reliable marker than I:T ratio ≥0.2 for fetal inflammatory response in newborns with combined thrombocytopenia and leukopenia during the initial assessment of intrauterine infection. Future prospective studies are required to confirm findings from this report.

Quantitative Classification of 3D Collagen Fiber Organization From Volumetric Images.

Collagen fibers in biological tissues have a complex 3D organization containing rich information linked to tissue mechanical properties and are affected by mutations that lead to diseases. Quantitative assessment of this 3D collagen fiber organization could help to develop reliable biomechanical models and understand tissue structure-function relationships, which impact diagnosis and treatment of diseases or injuries. While there are advanced techniques for imaging collagen fibers, published methods for quantifying 3D collagen fiber organization have been sparse and give limited structural information which cannot distinguish a wide range of 3D organizations. In this article, we demonstrate an algorithm for quantitative classification of 3D collagen fiber organization. The algorithm first simulates five groups, or classifications, of fiber organization: unidirectional, crimped, disordered, two-fiber family, and helical. These five groups are widespread in natural tissues and are known to affect the tissue's mechanical properties. We use quantitative metrics based on features such as preferred 3D fiber orientation and spherical variance to differentiate each classification in a repeatable manner. We validate our algorithm by applying it to second-harmonic generation images of collagen fibers in tendon and cervix tissue that has been sectioned in specified orientations, and we find strong agreement between classification from simulated data and the physical fiber organization. Our approach provides insight for interpreting 3D fiber organization directly from volumetric images. This algorithm could be applied to other fiber-like structures that are not necessarily made of collagen.

1D and 2D error assessment and correction for extrusion-based bioprinting using process sensing and control strategies.

The bioprinting literature currently lacks: (i) process sensing tools to measure material deposition, (ii) performance metrics to evaluate system performance, and (iii) control tools to correct for and avoid material deposition errors. The lack of process sensing tools limits in vivo functionality of bioprinted parts since accurate material deposition is critical to mimicking the heterogeneous structures of native tissues. We present a process monitoring and control strategy for extrusion-based fabrication that addresses all three gaps to improve material deposition. Our strategy uses a non-contact laser displacement scanner that measures both the spatial material placement and width of the deposited material. We developed a custom image processing script that uses the laser scanner data and defined error metrics for assessing material deposition. To implement process control, the script uses the error metrics to modify control inputs for the next deposition iteration in order to correct for the errors. A key contribution is the definition of a novel method to quantitatively evaluate the accuracy of printed constructs. We implement the process monitoring and control strategy on an extrusion-printing system to evaluate system performance and demonstrate improvement in both material placement and material width.

Cell seeding simulation on micropatterned islands shows cell density depends on area to perimeter ratio, not on island size or shape.

Protein micropatterned substrates have been used to control cell size, shape, and cell-cell contacts, characteristics that influence a range of cell behaviors from early cell differentiation to late stages of maturation. Knowing the initial island cell seeding density is important to interpreting results and understanding downstream cell behavior. While studies routinely report the intended or target cell seeding density, they do not report the actual cell seeding density on the islands. As cells proliferate, differences in initial cell seeding density could compound and may lead to misinterpretation of results. In this work, we present a cell seeding simulation and apply it to 100s of islands with a range of geometries (sizes and shapes) to explore how island cell seeding density relates to the target or unpatterned cell seeding density. We first experimentally validate the simulation and then show that normalized island cell seeding density depends on island size, shape, and spacing, but can be predicted solely from island area to perimeter ratio, A2P, via a power law relationship for a wide range of island geometries. Interestingly, normalized island cell seeding density is the same as the normalized unpatterned cell seeding density for A2P ≥ 17 µm. This simulation will help to design micropatterned substrates and to have more accurate representation of the island cell seeding density at the start of experiments. By knowing the island cell seeding density, we can more easily reproduce results across research groups to understand the roles of cell-cell contact and cell size and shape on cell behavior. STATEMENT OF SIGNIFICANCE: We present a cell seeding simulation on protein-micropatterned substrates and use it to simulate seeding across 100s of island geometries (size, shape, and spacing) covering two orders of magnitude in size. The simulation shows that island cell density varies significantly with island geometry compared to the target seeding density. However, island cell density can be predicted from one geometric parameter - the island's area to perimeter ratio. Results will help direct researchers on how to achieve uniform cell density across all island geometries. Since cell density and island shape both influence cell behaviors, such as differentiation, this simulation may help to isolate these factors, facilitate micropatterned substrate design, and provide a mechanism for more reproduceable results across studies.

An indentation-based approach to determine the elastic constants of soft anisotropic tissues.

Characterization of the mechanical properties of tissue can help to understand tissue mechanobiology, including disease diagnosis and progression. Indentation is increasingly used to measure the local mechanical properties of tissue, but it has not been fully adapted to capture anisotropic properties. This paper presents an indentation-based method to measure elastic constants of soft anisotropic tissues without additional mechanical tests. The approach uses measurement of the indentation modulus and the aspect ratio of the elliptical contact introduced by anisotropic mechanical properties of tissue to determine the elastic constants from finite element analysis. The imprinted area imparted by a fluorescent bead-coated spherical indenter showed the aspect ratio of the contact area, giving a generalized sense of the level of anisotropy, and instrumented indentation determined the indentation modulus. A parametric study using finite element simulation of the indentation tests established the relationship between the aspect ratio of contact and the non-dimensional ratios, Ex/Ey and Gxy/Ey; here, Ex and Ey are the Young's moduli (Ex > Ey) and Gxy is the shear modulus in the xy plane. For strongly anisotropic materials (Ex/Ey > 150), aspect ratio and indentation modulus are sufficient to determine Gxy and Ey. For weakly anisotropic materials, indentation modulus in the transverse direction, Ey, and the aspect ratio of contact in the anisotropic plane can be used to determine the elastic constants. The proposed approach improves the elastic characterization of soft, anisotropic biological materials from indentation and helps to elucidate the complex mechanical behavior of soft anisotropic tissues.

Direct process feedback in extrusion-based 3D bioprinting.

A major limitation in extrusion-based bioprinting is the lack of direct process control, which limits the accuracy and design complexity of printed constructs. The lack of direct process control results in a number of defects that can influence the functional and mechanical outcomes of the fabricated structures. The machine axes motion cannot be reliably used to predict material placement, and precise fabrication requires additional sensing of the material extrusion. We present an iteration-to-iteration process monitoring system that enables direct process control in the material deposition reference frame. To fabricate parts with low dimensional errors, we integrate a non-contact laser displacement scanner into the printing platform. After fabrication of the initial print using the as-designed reference trajectory, the laser scanner moves across the part to measure the material placement. A custom image processing algorithm compares the laser scanner data to the as-designed reference trajectory to generate an error vector. To compensate for the measured error, the algorithm modifies the axes reference trajectory for the second print iteration. We implement the in situ process monitoring and error compensation technique on an experimental platform to evaluate system performance and demonstrate improvement in spatial material placement.

Modulation of immune checkpoint molecule expression in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an aggressive B-cell malignancy for which novel therapeutics with improved efficacy are greatly needed. To provide support for clinical immune checkpoint blockade, we comprehensively evaluated the expression of therapeutically targetable immune checkpoint molecules on primary MCL cells. MCL cells showed constitutive expression of Programmed Death 1 (PD-1) and Programmed Death Ligand 1 (PD-L1), variable CD200, absent PD-L2, Lymphocyte Activation Gene 3 (LAG-3), and Cytotoxic T-cell Associated Protein 4 (CTLA-4). Effector cells from MCL patients expressed PD-1. Co-culture of MCL cells with T-cells induced PD-L1 surface expression, a phenomenon regulated by IFNγ and CD40:CD40L interaction. Induction of PD-L1 was attenuated by concurrent treatment with ibrutinib or duvelisib, suggesting BTK and PI3K are important mediators of PD-L1 expression. Overall, our data provide further insight into the expression of checkpoint molecules in MCL and support the use of PD-L1 blocking antibodies in MCL patients.