Synergistic Treatment of Breast Cancer by Combining the Antimicrobial Peptide Piscidin with a Modified Glycolipid.

Piscidin 3 (P3), a peptide produced by fish, and a hexyl ester-modified sophorolipid (SL-HE), have individually shown promise as antimicrobial and anticancer drugs. A recent report by our team revealed that combining P3 with SL-HE in a 1:8 molar ratio resulted in an 8-fold enhancement in peptide activity, while SL-HE improved by 25-fold its antimicrobial activity against the Gram-positive microorganism Bacillus cereus. Extending these findings, the same P3/SL-HE combination was assessed on two breast cancer cell lines: BT-474, a hormonally positive cell line, and MDA-MB-231, an aggressive triple-negative cell line. The results demonstrated that the 1:8 molar ratio of P3/SL-HE synergistically enhances the anticancer effects against both tumorigenic breast cell lines. Mechanistic studies indicate the activation of an intrinsic apoptotic cell death mechanism through an increase in reactive oxygen species and mitochondrial dysfunction and a secondary programmed necrotic pathway that involves pore formation in the plasma membrane. When a fibroblast cell line, CCD1065SK HDF, was utilized to determine selectivity, the synergistic SL-HE/P3 combination exhibited a protective property compared to the use of SL-HE alone and therefore afforded vastly improved selectivity indices. Given the promising results reported herein, the synergistic combination of P3/SL-HE constitutes a novel strategy that merits further study for the treatment of breast cancer.

DMT1-dependent endosome-mitochondria interactions regulate mitochondrial iron translocation and metastatic outgrowth.

Transient early endosome (EE)-mitochondria interactions can mediate mitochondrial iron translocation, but the associated mechanisms are still elusive. We showed that Divalent Metal Transporter 1 (DMT1) sustains mitochondrial iron translocation via EE-mitochondria interactions in triple-negative MDA-MB-231, but not in luminal A T47D breast cancer cells. DMT1 silencing increases labile iron pool (LIP) levels and activates PINK1/Parkin-dependent mitophagy in MDA-MB-231 cells. Mitochondrial bioenergetics and the iron-associated protein profile were altered by DMT1 silencing and rescued by DMT1 re-expression. Transcriptomic profiles upon DMT1 silencing are strikingly different between 2D and 3D culture conditions, suggesting that the environment context is crucial for the DMT1 knockout phenotype observed in MDA-MB-231 cells. Lastly, in vivo lung metastasis assay revealed that DMT1 silencing promoted the outgrowth of lung metastatic nodules in both human and murine models of triple-negative breast cancer cells. These findings reveal a DMT1-dependent pathway connecting EE-mitochondria interactions to mitochondrial iron translocation and metastatic fitness of breast cancer cells.

Molecularly Engineered Surfactin Analogues Induce Nonapoptotic-Like Cell Death and Increased Selectivity in Multiple Breast Cancer Cell Types.

Surfactin, a negatively charged amphiphilic lipopeptide biosurfactant, is synthesized by the bacterium Bacillus subtilis. It consists of a cyclic heptapeptide and an 11-15C ß-hydroxy fatty acid. To probe how the modification of the molecular skeleton of surfactin influences its selectivity and activity against breast cancer, six synthetic surfactins were generated. Modifications were accomplished by conjugating amine-functionalized molecules to the Glu and Asp carboxyl moieties of the heptapeptide. The resulting synthetic surfactins provided a diverse series of molecules with differences in charge, size, and hydrophilicity. After purification and structural analysis, insights into biological activity and specificity were generated for each compound. Dose-dependent growth inhibition was determined for four tumorigenic breast cancer cell lines in monolayer and spheroid morphologies, as well as nontumorigenic fibroblasts and sheep erythrocytes, which were utilized to determine selectivity indices. Results indicated that two compounds, which have amplified anionic charge, had increased activity on breast cancer, with reduced activity on nontumorigenic fibroblasts and erythrocytes. Cationic derivative surf-ethylenediamine has increased activity on all cell lines tested. Novel correlations between dose-response activities and physicochemical properties of all compounds determined that there is a significant correlation between the critical micelle concentration and activity against multiple cell lines.

Sophorolipid Candidates Demonstrate Cytotoxic Efficacy against 2D and 3D Breast Cancer Models.

Sophorolipids are biosurfactants derived from the nonpathogenic yeasts such as Starmerella bombicola with potential efficacy in anticancer applications. Simple and cost-effective synthesis of these drugs makes them a promising alternative to traditional chemotherapeutics, pending their success in preliminary drug-screening. Drug-screening typically utilizes 2D cell monolayers due to their simplicity and ease of high-throughput assessment. However, 2D assays fail to capture the complexity and 3D context of the tumor microenvironment and have consequently been implicated in the high percentage of drugs investigated in vitro that later fail in clinical trials. Herein, we screened two sophorolipid candidates and a clinically-used chemotherapeutic, doxorubicin, on in vitro breast cancer models ranging from 2D monolayers to 3D spheroids, employing optical coherence tomography to confirm these morphologies. We calculated corresponding IC50 values for these drugs and found one of the sophorolipids to have comparable toxicities to the chemotherapeutic control. Our findings show increased drug resistance associated with model dimensionality, such that all drugs tested showed that 3D spheroids exhibited higher IC50 values than their 2D counterparts. These findings demonstrate promising preliminary data to support the use of sophorolipids as a more affordable alternative to traditional clinical interventions and demonstrate the importance of 3D tumor models in assessing drug response.

Non-Destructive Evaluation of Regional Cell Density Within Tumor Aggregates Following Drug Treatment.

Multicellular tumor spheroid (MCTSs) models have demonstrated increasing utility for in vitro study of cancer progression and drug discovery. These relatively simple avascular constructs mimic key aspects of in vivo tumors, such as 3D structure and pathophysiological gradients. MCTSs models can provide insights into cancer cell behavior during spheroid development and in response to drugs; however, their requisite size drastically limits the tools used for non-destructive assessment. Optical Coherence Tomography structural imaging and Imaris 3D analysis software are explored for rapid, non-destructive, and label-free measurement of regional cell density within MCTSs. This approach is utilized to assess MCTSs over a 4-day maturation period and throughout an extended 5-day treatment with Trastuzumab, a clinically relevant anti-HER2 drug. Briefly, AU565 HER2+ breast cancer MCTSs were created via liquid overlay with or without the addition of Matrigel (a basement membrane matrix) to explore aggregates of different morphologies (thicker, disk-like 2.5D aggregates or flat 2D aggregates, respectively). Cell density within the outer region, transitional region, and inner core was characterized in matured MCTSs, revealing a cell-density gradient with higher cell densities in core regions compared to outer layers. The matrix addition redistributed cell density and enhanced this gradient, decreasing outer zone density and increasing cell compaction in the cores. Cell density was quantified following drug treatment (0 h, 24 h, 5 days) within progressively deeper 100 µm zones to assess potential regional differences in drug response. By the final timepoint, nearly all cell death appeared to be constrained to the outer 200 µm of each aggregate, while cells deeper in the aggregate appeared largely unaffected, illustrating regional differences in the drug response, possibly due to limitations in drug penetration. The current protocol provides a unique technique to non-destructively quantify regional cell density within dense cellular tissues and measure it longitudinally.

Sophorolipids: Anti-cancer activities and mechanisms.

Sophorolipids (SLs) are biosurfactants synthesized as secondary metabolites by non-pathogenic yeasts and other microorganisms. They are members of glycolipid microbial surfactant family that consists of a sophorose polar head group and, most often, an ω-1 hydroxylated fatty acid glycosidically linked to the sophorose moiety. Since the fermentative production of SLs is high (>200 g/L), SLs have the potential to provide low-cost therapeutics. Natural and modified SLs possess anti-cancer activity against a wide range of cancer cell lines such as those derived from breast, cervical, colon, liver, brain, and the pancreas. Corresponding data on their cytotoxicity against noncancerous cell lines including human embryo kidney, umbilical vein, and mouse fibroblasts is also discussed. These results are compiled to elucidate trends in SL-structures that lead to higher efficacy against cancer cell lines and lower cytotoxicity for normal cell lines. While extrapolation of these results provides some insights into the design of SLs with optimal therapeutic indices, we also provide a critical assessment of gaps and inconsistencies in the literature as well as the lack of data connecting structure-to-anticancer and cytotoxicity on normal cells. Furthermore, SL-mechanism of action against cancer cell lines, that includes proliferation inhibition, induction of apoptosis, membrane disruption and mitochondria mediated pathways are discussed. Perspectives on future research to develop SL anticancer therapeutics is discussed.

Non-Destructive Tumor Aggregate Morphology and Viability Quantification at Cellular Resolution, During Development and in Response to Drug.

Three-dimensional (3D) tissue-engineered in vitro models, particularly multicellular spheroids and organoids, have become important tools to explore disease progression and guide the development of novel therapeutic strategies. These avascular constructs are particularly powerful in oncological research due to their ability to mimic several key aspects of in vivo tumors, such as 3D structure and pathophysiologic gradients. Advancement of spheroid models requires characterization of critical features (i.e., size, shape, cellular density, and viability) during model development, and in response to treatment. However, evaluation of these characteristics longitudinally, quantitatively and non-invasively remains a challenge. Herein, Optical Coherence Tomography (OCT) is used as a label-free tool to assess 3D morphologies and cellular densities of tumor spheroids generated via the liquid overlay technique. We utilize this quantitative tool to assess Matrigel's influence on spheroid morphologic development, finding that the absence of Matrigel produces flattened, disk-like aggregates rather than 3D spheroids with physiologically-relevant features. Furthermore, this technology is adapted to quantify cell number within tumor spheroids, and to discern between live and dead cells, to non-destructively provide valuable information on tissue/construct viability, as well as a proof-of-concept for longitudinal drug efficacy studies. Together, these findings demonstrate OCT as a promising noninvasive, quantitative, label-free, longitudinal and cell-based method that can assess development and drug response in 3D cellular aggregates at a mesoscopic scale.

Laser-based 3D bioprinting for spatial and size control of tumor spheroids and embryoid bodies.

3D multicellular aggregates, and more advanced organotypic systems, have become central tools in recent years to study a wide variety of complex biological processes. Most notably, these model systems have become mainstream within oncology (multicellular tumor spheroids) and regenerative medicine (embryoid bodies) research. However, the biological behavior of these in vitro tissue surrogates is extremely sensitive to their aggregate size and geometry. Indeed, both of these geometrical parameters are key in producing pathophysiological gradients responsible for cellular and structural heterogeneity, replicating in vivo observations. Moreover, the fabrication techniques most widely used for producing these models lack the ability to accurately control cellular spatial location, an essential component for regulating homotypic and heterotypic cell signaling. Herein, we report on a 3D bioprinting technique, laser direct-write (LDW), that enables precise control of both spatial patterning and size of cell-encapsulating microbeads. The generated cell-laden beads are further processed into core-shelled structures, allowing for the growth and formation of self-contained, self-aggregating cells (e.g., breast cancer cells, embryonic stem cells). Within these structures we demonstrate our ability to produce multicellular tumor spheroids (MCTSs) and embryoid bodies (EBs) with well-controlled overall size and shape, that can be designed on demand. Furthermore, we investigated the impact of aggregate size on the uptake of a commonly employed ligand for receptor-mediated drug delivery, Transferrin, indicating that larger tumor spheroids exhibit greater spatial heterogeneity in ligand uptake. Taken together, these findings establish LDW as a versatile biomanufacturing platform for bioprinting and patterning core-shelled structures to generate size-controlled 3D multicellular aggregates. STATEMENT OF SIGNIFICANCE: Multicellular 3D aggregates are powerful in vitro models used to study a wide variety of complex biological processes, particularly within oncology and regenerative medicine. These tissue surrogates are fabricated using environments that encourage cellular self-assembly. However, specific applications require control of aggregate size and position to recapitulate key in vivo parameters (e.g., pathophysiological gradients and homotypic/heterotypic cell signaling). Herein, we demonstrate the ability to create and spatially pattern size-controlled embryoid bodies and tumor spheroids, using laser-based 3D bioprinting. Furthermore, we investigated the effect of tumor spheroid size on internalization of Transferrin, a common ligand for targeted therapy, finding greater spatial heterogeneity in our large aggregates. Overall, this technique offers incredible promise and flexibility for fabricating idealized 3D in vitro models.

The effect of engineered nanotopography of electrospun microfibers on fiber rigidity and macrophage cytokine production.

Currently, it is unknown how the mechanical properties of electrospun fibers, and the presentation of surface nanotopography influence macrophage gene expression and protein production. By further elucidating how specific fiber properties (mechanical properties or surface properties) alter macrophage behavior, it may be possible to create electrospun fiber scaffolds capable of initiating unique cellular and tissue responses. In this study, we determined the elastic modulus and rigidity of fibers with varying topographies created by finely controlling humidity and including a non-solvent during electrospinning. In total,five fiber scaffold types were produced. Analysis of fiber physical properties demonstrated no change in fiber diameter amongst the five different fiber groups. However, the four different fibrous scaffolds with nanopits or divots each possessed different numbers of pits with different nanoscale dimensions. Unpolarized bone marrow derived murine macrophages (M0), macrophages polarized towards a pro-inflammatory phenotype (M1), or macrophages polarized towards anti-inflammatory phenotype (M2b) were placed onto each of the scaffolds and cytokine RNA expression and protein production were analyzed. Specific nanotopographies did not appreciably alter cytokine production from undifferentiated macrophages (M0) or anti-inflammatory macrophages (M2b), but a specific fiber (with many small pits) did increase IL-12 transcript and IL-12 protein production compared to fibers with small divots. When analyzing the mechanical properties between fibers with divots or with many small pits,divoted fibers possessed similar elastic moduli but different stiffness values. In total,we present techniques capable of creating unique electrospun fibers. These unique fibers have varying fiber mechanical characteristics and modestly modulate macrophage cytokine expression.

A new experimental model for force enhancement: steady-state and transient observations of the Drosophila jump muscle.

The increase in steady-state force after active lengthening in skeletal muscle, termed force enhancement (FE), has been observed for nearly one century. Although demonstrated experimentally at various structural levels, the underlying mechanism(s) remain unknown. We recently showed that the Drosophila jump muscle is an ideal model for investigating mechanisms behind muscle physiological properties, because its mechanical characteristics, tested thus far, duplicate those of fast mammalian skeletal muscles, and Drosophila has the advantage that it can be more easily genetically modified. To determine if Drosophila would be appropriate to investigate FE, we performed classic FE experiments on this muscle. Steady-state FE (FESS), following active lengthening, increased by 3, 7, and 12% of maximum isometric force, with increasing stretch amplitudes of 5, 10, and 20% of optimal fiber length (FLOPT), yet was similar for stretches across increasing stretch velocities of 4, 20, and 200% FLOPT/s. These FESS characteristics of the Drosophila jump muscle closely mimic those observed previously. Jump muscles also displayed typical transient FE characteristics. The transient force relaxation following active stretch was fit with a double exponential, yielding two phases of force relaxation: a fast initial relaxation of force, followed by a slower recovery toward steady state. Our analyses identified a negative correlation between the slow relaxation rate and FESS, indicating that there is likely an active component contributing to FE, in addition to a passive component. Herein, we have established the Drosophila jump muscle as a new and genetically powerful experimental model to investigate the underlying mechanism(s) of FE.

Locked plate fixation of the comminuted distal fibula: a biomechanical study.

BACKGROUND: The purpose of this study was to compare the biomechanical properties of locked versus nonlocked lateral fibular bridge plating of comminuted, unstable ankle fractures in a mode of catastrophic failure. METHODS: We created comminuted Weber C fractures in 8 paired limbs from fresh cadavers. Fractures were plated with either standard or locked one-third tubular bridge plating techniques. Specimens were biomechanically evaluated by external rotation to failure while subjected to a compressive load approximating body weight. We measured the angle to failure, torque to failure, energy to failure and construct stiffness. RESULTS: There was no significant difference in construct stiffness or other biomechanical properties between locked and standard one-third tubular plating techniques. CONCLUSION: We found no difference in biomechanical properties between locked and standard bridge plating of a comminuted Weber C fibular fracture in a model of catastrophic failure. It is likely that augmentation of fixation with K-wires or transtibial screws provides a construct superior to locked bridge plating alone. Further biomechanical and clinical analysis is required to improve understanding of the role of locked plating in ankle fractures and in osteoporotic bone.

Gelatin-based laser direct-write technique for the precise spatial patterning of cells.

Laser direct-writing provides a method to pattern living cells in vitro, to study various cell-cell interactions, and to build cellular constructs. However, the materials typically used may limit its long-term application. By utilizing gelatin coatings on the print ribbon and growth surface, we developed a new approach for laser cell printing that overcomes the limitations of Matrigel™. Gelatin is free of growth factors and extraneous matrix components that may interfere with cellular processes under investigation. Gelatin-based laser direct-write was able to successfully pattern human dermal fibroblasts with high post-transfer viability (91% ± 3%) and no observed double-strand DNA damage. As seen with atomic force microscopy, gelatin offers a unique benefit in that it is present temporarily to allow cell transfer, but melts and is removed with incubation to reveal the desired application-specific growth surface. This provides unobstructed cellular growth after printing. Monitoring cell location after transfer, we show that melting and removal of gelatin does not affect cellular placement; cells maintained registry within 5.6 ± 2.5 µm to the initial pattern. This study demonstrates the effectiveness of gelatin in laser direct-writing to create spatially precise cell patterns with the potential for applications in tissue engineering, stem cell, and cancer research.

Laser-based direct-write techniques for cell printing.

Fabrication of cellular constructs with spatial control of cell location (+/-5 microm) is essential to the advancement of a wide range of applications including tissue engineering, stem cell and cancer research. Precise cell placement, especially of multiple cell types in co- or multi-cultures and in three dimensions, can enable research possibilities otherwise impossible, such as the cell-by-cell assembly of complex cellular constructs. Laser-based direct writing, a printing technique first utilized in electronics applications, has been adapted to transfer living cells and other biological materials (e.g., enzymes, proteins and bioceramics). Many different cell types have been printed using laser-based direct writing, and this technique offers significant improvements when compared to conventional cell patterning techniques. The predominance of work to date has not been in application of the technique, but rather focused on demonstrating the ability of direct writing to pattern living cells, in a spatially precise manner, while maintaining cellular viability. This paper reviews laser-based additive direct-write techniques for cell printing, and the various cell types successfully laser direct-written that have applications in tissue engineering, stem cell and cancer research are highlighted. A particular focus is paid to process dynamics modeling and process-induced cell injury during laser-based cell direct writing.

The mechanical properties of Drosophila jump muscle expressing wild-type and embryonic Myosin isoforms.

Transgenic Drosophila are highly useful for structure-function studies of muscle proteins. However, our ability to mechanically analyze transgenically expressed mutant proteins in Drosophila muscles has been limited to the skinned indirect flight muscle preparation. We have developed a new muscle preparation using the Drosophila tergal depressor of the trochanter (TDT or jump) muscle that increases our experimental repertoire to include maximum shortening velocity (V(slack)), force-velocity curves and steady-state power generation; experiments not possible using indirect flight muscle fibers. When transgenically expressing its wild-type myosin isoform (Tr-WT) the TDT is equivalent to a very fast vertebrate muscle. TDT has a V(slack) equal to 6.1 +/- 0.3 ML/s at 15 degrees C, a steep tension-pCa curve, isometric tension of 37 +/- 3 mN/mm(2), and maximum power production at 26% of isometric tension. Transgenically expressing an embryonic myosin isoform in the TDT muscle increased isometric tension 1.4-fold, but decreased V(slack) 50% resulting in no significant difference in maximum power production compared to Tr-WT. Drosophila expressing embryonic myosin jumped <50% as far as Tr-WT that, along with comparisons to frog jump muscle studies, suggests fast muscle shortening velocity is relatively more important than high tension generation for Drosophila jumping.

CD11b+ neutrophils predominate over RAM11+ macrophages in stretch-injured muscle.

The purpose of this study was to determine whether both neutrophils and macrophages infiltrate the hematoma site of stretch-injured rabbit tibialis anterior muscle. The Mab.198 antibody was used to detect CD11b(+) neutrophils or macrophages. Neutrophils were identified specifically by using the RPN3/57 antibody. The RAM11 antibody was used to detect macrophages. The histological characteristics of the hematoma site, torn fibers or inflammatory cells, were present primarily at 4 and 24 h, but not at 48 and 72 h after injury. A difference in the Mab.198(+) cellular concentration was detected over time between uninjured and injured muscles (P = 0.03). The injured-uninjured difference in the RPN3/57(+) or RAM11(+) cellular concentrations approached significance (P = 0.07) or else was deemed insignificant (P = 0.13), respectively. Therefore, neutrophils may predominate over RAM11(+) macrophages in stretch-injured muscle. These findings may influence the antiinflammatory strategies used to treat stretch injuries.