Cellular and molecular bioengineering最新文献

Introduction: In the colorectal cancer (CRC) tumor microenvironment, cancerous and precancerous cells continuously experience mechanical forces associated with peristalsis. Given that mechanical forces like shear stress and strain can positively impact cancer progression, we explored the hypothesis that peristalsis may also contribute to malignant progression in CRC. We defined malignant progression as enrichment of cancer stem cells and the acquisition of invasive behaviors, both vital to CRC progression.

Methods: We leveraged our peristalsis bioreactor to expose CRC cell lines (HCT116), patient-derived xenograft (PDX1,2) lines, or non-cancerous intestinal cells (HIEC-6) to forces associated with peristalsis in vitro. Cells were maintained in static control conditions or exposed to peristalsis for 24 h prior to assessment of cancer stem cell (CSC) emergence or the acquisition of invasive phenotypes.

Results: Exposure of HCT116 cells to peristalsis significantly increased the emergence of LGR5⁺ CSCs by 1.8-fold compared to static controls. Peristalsis enriched LGR5 positivity in several CRC cell lines, notably significant in KRAS mutant lines. In contrast, peristalsis failed to increase LGR5⁺ in non-cancerous intestinal cells, HIEC-6. LGR5⁺ emergence downstream of peristalsis was dependent on ROCK and Wnt activity, and not YAP1 activation. Additionally, HCT116 cells adopted invasive morphologies when exposed to peristalsis, with increased filopodia density and epithelial to mesenchymal gene expression, in a Wnt dependent manner.

Conclusions: Peristalsis associated forces drive malignant progression of CRC via ROCK, YAP1, and Wnt-related mechanotransduction.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-023-00776-w.

Introduction: While most patients with triple negative breast cancer receive radiation therapy to improve outcomes, a significant subset of patients continue to experience recurrence. Macrophage infiltration into radiation-damaged sites has been shown to promote breast cancer recurrence in pre-clinical models. However, the mechanisms that drive recurrence are unknown. Here, we developed a novel spheroid model to evaluate macrophage-mediated tumor cell recruitment.

Methods: We characterized infiltrating macrophage phenotypes into irradiated mouse mammary tissue via flow cytometry. We then engineered a spheroid model of radiation damage with primary fibroblasts, macrophages, and 4T1 mouse mammary carcinoma cells using in vivo macrophage infiltration results to inform our model. We analyzed 4T1 infiltration into spheroids when co-cultured with biologically relevant ratios of pro-healing M2:pro-inflammatory M1 macrophages. Finally, we quantified interleukin 6 (IL-6) secretion associated with conditions favorable to tumor cell infiltration, and we directly evaluated the impact of IL-6 on tumor cell invasiveness in vitro and in vivo.

Results: In our in vivo model, we observed a significant increase in M2 macrophages in mouse mammary glands 10 days post-irradiation. We determined that tumor cell motility toward irradiated spheroids was enhanced in the presence of a 2:1 ratio of M2:M1 macrophages. We also measured a significant increase in IL-6 secretion after irradiation both in vivo and in our model. This secretion increased tumor cell invasiveness, and tumor cell invasion and recruitment were mitigated by neutralizing IL-6.

Conclusions: Our work suggests that interactions between infiltrating macrophages and damaged stromal cells facilitate breast cancer recurrence through IL-6 signaling.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-023-00775-x.

Introduction: Cell proliferation represents a major hallmark of cancer biology, and manifests itself in the assessment of tumor growth, drug resistance and metastasis. Tracking cell proliferation or cell fate at the single-cell level can reveal phenotypic heterogeneity. However, characterization of cell proliferation is typically done in bulk assays which does not inform on cells that can proliferate under given environmental perturbations. Thus, there is a need for single-cell approaches that allow longitudinal tracking of the fate of a large number of individual cells to reveal diverse phenotypes.

Methods: We fabricated a new microfluidic architecture for high efficiency capture of single tumor cells, with the capacity to monitor cell divisions across multiple daughter cells. This single-cell proliferation (SCP) device enabled the quantification of the fate of more than 1000 individual cancer cells longitudinally, allowing comprehensive profiling of the phenotypic heterogeneity that would be otherwise masked in standard cell proliferation assays. We characterized the efficiency of single cell capture and demonstrated the utility of the SCP device by exposing MCF-7 breast tumor cells to different doses of the chemotherapeutic agent doxorubicin.

Results: The single cell trapping efficiency of the SCP device was found to be ~ 85%. At the low doses of doxorubicin (0.01 µM, 0.001 µM, 0.0001 µM), we observed that 50-80% of the drug-treated cells had undergone proliferation, and less than 10% of the cells do not proliferate. Additionally, we demonstrated the potential of the SCP device in circulating tumor cell applications where minimizing target cell loss is critical. We showed selective capture of breast tumor cells from a binary mixture of cells (tumor cells and white blood cells) that was isolated from blood processing. We successfully characterized the proliferation statistics of these captured cells despite their extremely low counts in the original binary suspension.

Conclusions: The SCP device has significant potential for cancer research with the ability to quantify proliferation statistics of individual tumor cells, opening new avenues of investigation ranging from evaluating drug resistance of anti-cancer compounds to monitoring the replicative potential of patient-derived cells.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-023-00773-z.

Purpose: The importance of mechanical forces and microenvironment in guiding cellular behavior has been widely accepted. Together with the extracellular matrix (ECM), epithelial cells form a highly connected mechanical system subjected to various mechanical cues from their environment, such as ECM stiffness, and tensile and compressive forces. ECM stiffness has been linked to many pathologies, including tumor formation. However, our understanding of the effect of ECM stiffness and its heterogeneities on rapid force transduction in multicellular systems has not been fully addressed.

Methods: We used experimental and computational methods. Epithelial cells were cultured on elastic hydrogels with fluorescent nanoparticles. Single cells were moved by a micromanipulator, and epithelium and substrate deformation were recorded. We developed a computational model to replicate our experiments and quantify the force distribution in the epithelium. Our model further enabled simulations with local stiffness gradients.

Results: We found that substrate stiffness affects the force transduction and the cellular deformation following an external force. Also, our results indicate that the heterogeneities, e.g., gradients, in the stiffness can substantially influence the strain redistribution in the cell monolayers. Furthermore, we found that the cells' apico-basal elasticity provides a level of mechanical isolation between the apical cell-cell junctions and the basal focal adhesions.

Conclusions: Our simulation results show that increased ECM stiffness, e.g., due to a tumor, can mechanically isolate cells and modulate rapid mechanical signaling between cells over distances. Furthermore, the developed model has the potential to facilitate future studies on the interactions between epithelial monolayers and elastic substrates.

Supplementary information: The online version of this article (10.1007/s12195-023-00772-0) contains supplementary material, which is available to authorized users.

Introduction: Abnormal angiogenesis is central to vascular disease and cancer, and noninvasive biomarkers of vascular origin are needed to evaluate patients and therapies. Vascular endothelial growth factor receptors (VEGFRs) are often dysregulated in these diseases, making them promising biomarkers, but the need for an invasive biopsy has limited biomarker research on VEGFRs. Here, we pioneer a blood biopsy approach to quantify VEGFR plasma membrane localization on two circulating vascular proxies: circulating endothelial cells (cECs) and circulating progenitor cells (cPCs).

Methods: Using quantitative flow cytometry, we examined VEGFR expression on cECs and cPCs in four age-sex groups: peri/premenopausal females (aged < 50 years), menopausal/postmenopausal females (≥ 50 years), and younger and older males with the same age cut-off (50 years).

Results: cECs in peri/premenopausal females consisted of two VEGFR populations: VEGFR-low (~ 55% of population: population medians ~ 3000 VEGFR1 and 3000 VEGFR2/cell) and VEGFR-high (~ 45%: 138,000 VEGFR1 and 39,000-236,000 VEGFR2/cell), while the menopausal/postmenopausal group only possessed the VEGFR-low cEC population; and 27% of cECs in males exhibited high plasma membrane VEGFR expression (206,000 VEGFR1 and 155,000 VEGFR2/cell). The absence of VEGFR-high cEC subpopulations in menopausal/postmenopausal females suggests that their high-VEGFR cECs are associated with menstruation and could be noninvasive proxies for studying the intersection of age-sex in angiogenesis. VEGFR1 plasma membrane localization in cPCs was detected only in menopausal/postmenopausal females, suggesting a menopause-specific regenerative mechanism.

Conclusions: Overall, our quantitative, noninvasive approach targeting cECs and cPCs has provided the first insights into how sex and age influence VEGFR plasma membrane localization in vascular cells.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-023-00771-1.

Introduction: Biomaterials that enable in situ recruitment and modulation of immune cells have demonstrated tremendous promise for developing potent cancer immunotherapy such as therapeutic cancer vaccine. One challenge related to biomaterial scaffold-based cancer vaccines is the development of macroporous materials that are biocompatible and stable, enable controlled release of chemokines to actively recruit a large number of dendritic cells (DCs), contain macropores that are large enough to home the recruited DCs, and support the survival and proliferation of DCs.

Methods: Bio-adhesive macroporous gelatin hydrogels were synthesized and characterized for mechanical properties, porous structure, and adhesion towards tissues. The recruitment of immune cells including DCs to chemokine-loaded bioadhesive macroporous gels was analyzed. The ability of gels loaded with granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor extracellular vesicles (EVs) to elicit tumor-specific CD8⁺ T cell responses was also analyzed.

Results: Here we develop a bioadhesive macroporous hydrogel that can strongly adhere to tissues, contain macropores that are large enough to home immune cells, are mechanically tough, and enable controlled release of chemokines to recruit and modulate immune cells in situ. The macroporous hydrogel is composed of a double crosslinked network of gelatin and polyacrylic acid, and the macropores are introduced via cryo-polymerization. By incorporating GM-CSF and tumor EVs into the macroporous hydrogel, a high number of DCs can be recruited in situ to process and present EV-encased antigens. These tumor antigen-presenting DCs can then traffic to lymphatic tissues to prime antigen-specific CD8⁺ T cells.

Conclusion: This bioadhesive macroporous hydrogel system provides a new platform for in situ recruitment and modulation of DCs and the development of enhanced immunotherapies including tumor EV vaccines. We also envision the promise of this material system for drug delivery, tissue regeneration, long-term immunosuppression, and many other applications.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-023-00770-2.

Introduction: Pediatric subglottic stenosis (SGS) results from prolonged intubation where scar tissue leads to airway narrowing that requires invasive surgery. We have recently discovered that modulating the laryngotracheal microbiome can prevent SGS. Herein, we show how our patent-pending antimicrobial peptide-eluting endotracheal tube (AMP-ET) effectively modulates the local airway microbiota resulting in reduced inflammation and stenosis resolution.

Materials and methods: We fabricated mouse-sized ETs coated with a polymeric AMP-eluting layer, quantified AMP release over 10 days, and validated bactericidal activity for both planktonic and biofilm-resident bacteria against Staphylococcus aureus and Pseudomonas aeruginosa. Ex vivo testing: we inserted AMP-ETs and ET controls into excised laryngotracheal complexes (LTCs) of C57BL/6 mice and assessed biofilm formation after 24 h. In vivo testing: AMP-ETs and ET controls were inserted in sham or SGS-induced LTCs, which were then implanted subcutaneously in receptor mice, and assessed for immune response and SGS severity after 7 days.

Results: We achieved reproducible, linear AMP release at 1.16 µg/day resulting in strong bacterial inhibition in vitro and ex vivo. In vivo, SGS-induced LTCs exhibited a thickened scar tissue typical of stenosis, while the use of AMP-ETs abrogated stenosis. Notably, SGS airways exhibited high infiltration of T cells and macrophages, which was reversed with AMP-ET treatment. This suggests that by modulating the microbiome, AMP-ETs reduce macrophage activation and antigen specific T cell responses resolving stenosis progression.

Conclusion: We developed an AMP-ET platform that reduces T cell and macrophage responses and reduces SGS in vivo via airway microbiome modulation.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-023-00769-9.

[This corrects the article DOI: 10.1007/s12195-023-00767-x.].

Promotion and tenure (P&T) remain the central tenets of academia. The criteria for P&T both create and reflect the mission of an institution. The discipline of biomedical engineering is built upon the invention and translation of tools to address unmet clinical needs. 'Broadening the bar' for P&T to include efforts in innovation, entrepreneurship, and technology-based transfer (I/E/T) will require establishing the criteria and communication of methodology for their evaluation. We surveyed the department chairs across the fields of biomedical and bioengineering to understand the state-of-the-art in incorporation, evaluation, and definition of I/E/T as applied to the P&T process. The survey results reflected a commitment to increasing and respecting I/E/T activities as part of the P&T criteria. This was balanced by an equally strong desire for improving the education and policy for evaluating I/E/T internally as well as externally. The potential for 'broadening the bar' for P&T to include I/E/T activities in biomedical engineering may serve as an example for other fields in engineering and applied sciences, and a template for potential inclusion of additional efforts such as diversity, equity, and inclusion (DEI) into the pillars of scholarship, education, and service.