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Published in final edited form as: Immunobiology. 2023 Sep 13;228(6):152744. doi: 10.1016/j.imbio.2023.152744

Fluid shear stress enhances dendritic cell activation

Jenna A Dombroski 1, Schyler J Rowland 1, Abigail R Fabiano 1, Samantha V Knoblauch 1, Jacob M Hope 1, Michael R King 1,*
PMCID: PMC10841200  NIHMSID: NIHMS1933132  PMID: 37729773

Abstract

Ex vivo activation of dendritic cells has been widely explored for targeted therapies, although these treatments remain expensive. Reducing treatment costs while enhancing cell activation could help to make immunotherapies more accessible. Cells can be activated by both internal and external forces including fluid shear stress (FSS). FSS activates cells via opening of mechanosensitive ion channels. In this study, dendritic cells were activated by sustained exposure to circulatory levels of fluid shear stress using a cone-and-plate flow device and analyzed for activation markers. After 1 h of shear stress exposure, an increase in cytokine release was present in immortalized cells as well as phosphorylation of the proteins NF-κB and cFos in primary DCs. Changes in DC morphology, metabolism and proliferation were also observed. These compelling new findings point to the potential for using FSS to activate DCs FSS for ex vivo therapeutics.

Keywords: Cancer immunotherapy, dendritic cells, fluid shear stress

INTRODUCTION

Therapeutics have been developed in which a patient’s dendritic cells (DCs) are removed, activated ex vivo, and reintroduced into the body to enhance the efficacy of treatments.1–3 The first FDA-approved therapeutic cancer vaccine, Sipuleucel-T (Provenge), was approved in 2010 for the treatment of metastatic castration-resistant prostate cancer (mCRPC).4,5 For this therapy, DCs are activated with granulocyte macrophage-colony stimulating factor (GM-CSF) or prostatic acid phosphatase (PAP), facilitating T cell priming and enabling the targeting of prostate cancer cells.4,5 While Sipuleucel-T is associated with low treatment periods as well as low hospitalization rate due to adverse events, the cost of the therapeutic remains expensive to patients compared to traditional cancer treatments.4 Additionally, although a novel concept and showing promise when compared to placebo, Sipuleucel-T has demonstrated limited effectiveness in recent studies.6,7 Therefore, boosting the efficacy of Sipuleucel-T or finding alternative ex vivo therapeutics has emerged as a significant need.

Internal and external forces play roles in activating a variety of cell types, ranging from endothelial cells and immune cells to cancer cells.8–10 Shear stress is a force which activates cells by initiating membrane deformation, resulting in the opening of mechanosensitive ion channels (MSCs), which affect signal transduction through the influx of calcium ions.11–16 Calcium is a ubiquitous secondary messenger responsible for a host of intracellular responses.10,17–20 Fluid shear stress (FSS) is a force which immune cells experience due to blood flow in circulation.21–23 Several studies have demonstrated that blood cells such as T cells and platelets are sensitive to shear stress, with increased proliferation and/or cytokine release.13,24,25 Neutrophils have also been observed to have increased activation following FSS.26 Specifically, shear stress promotes the maturation, growth, and progression of the cell cycle in DCs.27 One study applied cyclic strain to DCs and observed a resulting increase in the expression of MHC II and costimulatory molecules, although there is a general dearth of research into DC activation via shear stress.28

FSS can be recreated in vitro within cone-and-plate or other flow devices.29–31 Cone-and-plate flow devices are advantageous in that, based on their designs, they apply the same local shear rate to cells at every location within the device, which is not true in pressure-driven flow or Couette flow devices.29 In this study, we stimulated DCs ex vivo with FSS applied via a cone-and-plate flow device, analyzing various markers of activation. Both immortalized DCs and two primary cell lines were used in this study.

RESULTS

FSS from cone-and-plate flow device does not significantly affect cell viability of DCs

DCs were suspended in complete media and stimulated with FSS in a cone-and-plate flow viscometer at 5 dyn/cm2 for 1 h. Tubes were left on a rotator for “static” conditions (<0.5 dyn.cm2) (Supplemental Figure 4A). The cone-and-plate viscometer creates an environment where the same shear rate is applied to all DCs regardless of their position within the volume (Fig. 1A). Following FSS exposure, cells were analyzed for viability using an Annexin V assay. DC2.4 cells under static conditions showed an average viability of 90.9% and cells under shear conditions showed an average viability of 83.8%. There was not a significant difference in viability in DCs under static conditions compared shear conditions (Fig. 1B–C). These results indicate that FSS was not causing significant apoptosis or necrosis in DCs and that experiments for ex vivo activation was justified.

Figure 1.

Figure 1.

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Cone-and-plate viscometer setup and cell viability assay. (A) Experimental design for FSS application. (B) Summary data for percent viability as determined by Annexin V assay. The average percent cell viability for DC2.4 cells under static conditions was 90.9 ± 1.9%, and 83.8 ± 6.2% for shear. There was no significant difference in these averages. (C) Graphical representation for interpreting Annexin V flow cytometry plots. Cells negative for propidium iodide (PI) and Annexin V (AV) were considered viable. Representative flow cytometry plots correspond to static and shear conditions.

Potent stimulator LPS plays a role in early MHC and costimulatory molecule expression on DCs following FSS

Following 1 h stimulation in a cone-and-plate flow device at 5 dyn/cm2 FSS, DC2.4 cells were plated and incubated at 37°C for 24 h to measure expression of later markers of activation and compared to static conditions. Expression of major histocompatibility complex (MHC) and costimulatory molecules including CD40, CD70, CD80, and CD86 are indicative of DCs functioning as antigen-presenting cells (APCs) to facilitate T cell priming.32–34 After 24 h, cells were analyzed for the presence of MHCs and costimulatory molecules. No significant increase in MHC expression with DC stimulation via FSS was observed (Fig. 2A). Costimulatory molecule expression was found to be enhanced in the presence of LPS, regardless of whether shear stress was present (Fig. 2B). This trend was observed for CD86, and significant for CD40 and CD80. The mean percentage expression of CD40 under static conditions was 3.8% without LPS, and 47% with LPS. Under shear conditions, a similar pattern was observed, with the mean percentage expression 14% without LPS and 45% with LPS.

Figure 2.

Figure 2.

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Costimulatory molecule and cytokine analysis of FSS-activated DCs. (A) Histogram reflecting representative data for MHC I expression following FSS and summary data for percentage of MHC I and MHC II expression. (B) Summary data for costimulatory molecule expression analyzed after 24 h and representative flow cytometry plot corresponding to static, static + LPS, shear, and shear + LPS conditions. A trend was evident in increased CD80 expression in the presence of LPS and a significant increase in CD40 expression in the presence of LPS. (C) Proteome profiler representative cytokine array results and corresponding cytokines. (D) Summary of cytokine release analyzed after 24 h. A significant increase in cytokine release was observed after DCs were stimulated via FSS compared to DCs under static conditions. *p<0.05, **p<0.01, ***p<0.005, and ****p<0.001.

Increase in cytokine release was observed after FSS stimulation

DC2.4 cells were plated for 24 h following stimulation via 5 dyn/cm2 FSS for 1 h, while control samples were maintained at 0 dyn/cm2 in a stationary viscometer (Supplemental Figure 4B). Cytokines and chemokines are a direct measure of immune cell activation, especially for DCs, where they aid in trafficking and driving specific T cell activation.35 A complete cytokine array was then performed using Mouse Cytokine Array Panel A, a proteome profiler from R&D Systems. 40 different cytokines were analyzed using the panel, comparing the supernatant of FSS-stimulated DC2.4 cells. There was an increase in cytokine release for TIMP-1, CXCL10, G-CSF, IL-6, M-CSF, CCL2, CCL4, CCL5, CXCL12, IL-27 and IL-1ra, and a significant increase for TNF-α, ICAM-1, CCL3 and CXCL2 (Fig. 2C–D). An analysis was performed to validate that BSA pre-blocking of the viscometers was not responsible for the activation seen during FSS application by blocking with MSA (Supplemental Figure 1A–B).

DCs stimulated by FSS maintain activation state for an extended period

Interestingly, while there were small differences observed in costimulatory molecule expression after 24 h, these differences were more prominent after 48 h. DCs 48 h after FSS treatment showed a trend of increased CD40, CD80 and CD86 expression compared to unstimulated cells (Fig. 3A). After 48 h, cytokine release, as detected via proteome profiler, was reduced from 24 h but still higher for most cytokines for FSS-stimulated cells (Fig. 3B).

Figure 3.

Figure 3.

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DC activation for extended time points. (A) Representative flow cytometry plot for CD40 expression after 48h. A trend of increased costimulatory molecule expression was observed for up to 48 h following FSS. (B) Summary data for cytokine release as observed after 48 h using proteome profiler to measure mean pixel density of array.

Changes in DC metabolism were evident following FSS

Increased uptake of 2-NBDG was detected for cells exposed to 5 dyn/cm2 of FSS for 1 h, compared to cells exposed to 0 dyn/cm2 (Fig. 4A). As 2-NBDG consists of a d-glucose analog, higher levels of 2-NBDG are indicative of increased glucose uptake by cells.36 Following FSS, notable visual changes in cell culture media were observed when compared to cells exposed to 0 dyn/cm2 (Supplemental Figure 2). Therefore, using a Thermo Scientific Orion Star A211 Benchtop Meter and pH Electrode Set, changes in pH were analyzed following FSS treatment. There was a significant increase in pH immediately following 1 h of FSS (Fig. 4B). Percent positive DCs with cell proliferation marker Ki67 was significantly increased immediately following FSS (Fig. 4C–D). These changes were still observed up to 24 h when compared to cells under static conditions (Fig. 4E–F).

Figure 4.

Figure 4.

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Metabolic and morphological changes to DCs following FSS treatment. (A) Glucose uptake by DC2.4 cells was increased following 5 dyn/cm2 of FSS exposure, as observed by the increase in 2-NBDG uptake into cells. (B) The pH of the media containing cells exposed to 5 dyn/cm2 FSS for 1 h became significantly higher compared to cells exposed to 0 dyn/cm2. (C) Representative flow cytometry plots of percentage of Ki67 expression following FSS. (D) Combined percentage Ki67 expression comparing cells under four conditions: static, static + LPS, shear, and shear + LPS. (E) Representative flow cytometry plots for percentage Ki67 expression over time in cells following FSS. (F) Percentage Ki67 expression over time in DCs following FSS. (G) RAC1 representative flow cytometry histogram. (H) RAC1 expression in DC2.4 cells following 1 h FSS. (I) Representative micrograph of dendrite formation in DCs stimulated with FSS. (J) Percent of DCs with dendrite formation following FSS stimulation. *p<0.05, **p<0.01, and ***p<0.005. Scale bar = 50 μm.

DC morphology was significantly affected by FSS with enhanced dendrite formation

Enhanced RAC1 is indicative of dendrite formation in DCs.37 Percent positive RAC1 expression in DC2.4 cells was significantly increased following FSS, compared to cells under static conditions (Fig. 4G–H). Additionally, the percentage of DCs with dendrite formation was significantly increased following stimulation via FSS, as observed via confocal microscopy (Fig. 4I–J). Actin levels were decreased following FSS treatment (Supplemental Figure 3).

Primary DCs become activated by FSS ex vivo

Primary cells were then investigated for activation via FSS. Nuclear factor-kappa B (NF-κB) phosphorylation was measured in primary DCs to analyze activation. NF-κB transcription factor is regulated by calcium influx, and leads to increased cytokine expression, maturation, survival, and cell cycle progression in a variety of cell types including DCs.38–42 Activator protein 1 (AP-1) expression was measured by analyzing activation of the AP-1 transcription factor complex using an antibody for phospho-c-Fos. AP-1 is another transcription factor that is regulated by calcium ions and responsible for controlling cellular processes such as proliferation and regulation of cytokine release.43,44 BMDCs were isolated from healthy mice and stimulated with 0 or 5 dyn/cm2 FSS for 1 h, as in experiments with immortalized cells. A trend in increased costimulatory molecule expression of BMDCs was observed (Fig. 5A). There was a significant increase in NF-κB and cFos phosphorylation (Fig. 5B–C). A heterogeneous population of myeloid and plasmacytoid DCs (mDCs and pDCs) isolated from healthy human volunteers were stimulated with 0 or 5 dyn/cm2 FSS for 1 h. Following FSS, a significant increase in NF-κB phosphorylation was observed, as well as a trend in enhanced CD86 (Fig. 5D–F).

Figure 5.

Figure 5.

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Primary DC response to stimulation via FSS. (A) Trend in increased costimulatory molecule expression of murine BMDCs exposed to 5 dyn/cm2 of FSS for 1 h, compared to cells exposed to 0 dyn/cm2. (B) Representative flow plots of BMDC NF-kB activation. (C) Significant increase in phosphorylation of NF-κB and cFos for BMDCs stimulated with FSS for 1 h. (D) Representative flow plots of NF-kB activation of primary human moDCs. (E) Increased NF-κB phosphorylation for primary human pDCs and mDCs following FSS stimulation for 1 h at 5 dyn/cm2, compared to cells exposed to 0 dyn/cm2. (F) Trend in increased CD86 costimulatory molecule in human DCs following FSS stimulation. *p<0.05.

DISCUSSION

To the best of our knowledge, this is the first study to demonstrate that FSS activates DCs. It was first established that shear stress minimally reduces cell viability in DCs, suggesting that it is a safe mechanism for ex vivo activation of immune cells. With just 1 h of applied FSS, significant cellular changes were recorded, indicative of DC activation.

Cytokine release and costimulatory molecule expression, two essential components of the immune synapse for T cell activation by APCs, were enhanced following FSS. DC cytokine release is important for recruiting immune cells and driving specific T cell responses35. While there was not a significant difference in MHC and costimulatory molecule expression in DCs following 24 h of FSS stimulation, there was a significant enhancement in the presence of the potent stimulator LPS, particularly for CD40. The results suggest that, if enhanced costimulatory molecule expression is desired at such an early time point, a stimulator may be necessary in addition to the ex vivo FSS treatments. Incubation with a tumor-specific antigen (TSA) or tumor-associated antigen (TAA) could aid in achieving this specific response; for instance, PAP could be applied to mCRPC therapies45. Nevertheless, at later time points, enhanced costimulatory molecule expression due to FSS alone was observed.

Exploring metabolic and morphological changes to DCs following FSS stimulation revealed interesting findings compared to non-stimulated cells. Cells showed increased glucose uptake and Ki67 expression and pH levels were significantly enhanced, indicating metabolic processes occurring at a cellular level. It would be interesting to further explore these aspects of DC function following FSS activation to gain further insight into the processes taking place. Additionally, changes in morphology with RAC1 and dendrite formation were observed, indicating DC activation as a successful APC with these conformational changes.

In primary cells, NF-𝜅B and cFos phosphorylation were enhanced with stimulation via FSS. Increased expression of these transcription factors gives insight into the pathways involved in DC activation via FSS. These transcription factors play a major role in DC function and maturation, particularly through the release of cytokines to drive T cell priming.

The activation observed throughout this study is likely a result of applied FSS activating mechanosensitive ion channels such as Piezo1. Further experiments using a Piezo1 inhibitor, such as GsMTx4 which inhibits cationic MSCs, could elucidate this mechanism. Although DCs in circulation are exposed to similar levels of FSS at specific time points in vivo, this method of ex vivo activation boasts the advantage of applying a continuous exposure at a constant shear stress for all cells within the apparatus. It would be interesting to explore the impact of spatially varying shear rate (such as in pressure-driven flow through a tube) and duration of shear, even oscillatory signals. Additionally, exploring responses of other types of DCs such as purified conventional DCs (cDCs) and monocyte DCs (moDCs) could inform how primary human DCs respond to FSS.

Despite the major advances in ex vivo therapeutics, the costs of these treatments remain high for patients. Identifying a way to reduce these costs while further enhancing activation will be advantageous for developing improved therapies. The results from this study suggest that ex vivo applied FSS could be used in the production of DC-based immunotherapies to drive down the costs of these treatments. Through the ex vivo stimulation of DCs via FSS in combination with an additional activating factor, the necessary transcription factors can be activated, cytokines released, and costimulatory molecules enhanced, to yield successful therapeutics.

MATERIALS AND METHODS

Cell culture:

DC2.4 murine DCs were purchased from Sigma-Aldrich (Catalog No. SCC142). DCs were grown in culture media consisting of RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 1X L-Glutamine, 1X non-essential amino acids, 1X HEPES buffer solution and 0.0054X β-Mercaptoethanol, as recommended by the manufacturer. Cells were maintained in an incubator at 37°C with 5% CO2, and experiments performed at ~80% confluency.

Reagents:

RPMI-1640 and FBS (Invitrogen), non-essential amino acids and HEPES buffer (Gibco), and β-Mercaptoethanol (Sigma-Aldrich) were obtained for preparation of the DC2.4 cell culture media. Hank’s balanced salt solution (HBSS) with and without calcium and magnesium was purchased from Gibco. Lipopolysaccharide (LPS) solution (500X) was purchased from Thermo Scientific. 32% paraformaldehyde aqueous solution, electron microscopy grade was obtained from Electron Microscopy Sciences. Anti-mouse PE-MHC (Class I H-2Kk) was purchased from Antibodies-Online.com. Anti-mouse PE-CD40 (5C3), anti-human PE-CD40 (5C3), and PE anti-CD86 human monoclonal antibody were purchased from BioLegend. Anti-mouse PE-CD70 (FR70), PE-CD80 (16–10A1), PE-I A/I E (M5/114.15.2) (MHC II), PE-IL-6 (MP5 20F3), PE-IL-12 (p40/p70) (C15.6), and PE mouse anti-Ki-67 (567720) were used for extracellular and intracellular staining (BD). Anti-mouse PE-CD83 (Michel 17), PE-CD197 (CCR7) (4B12), PE-phospho-NFkB p65 (Ser529) (NFkBp65S529-H3), PE-phospho-c-Fos (AP-1) (Ser32) (cFosS32-BA9), and PE (MA5–36891) were obtained from Thermo Scientific. Proteome Profiler Mouse Cytokine Array Kit, Panel A (ARY006) was used to analyze cytokine release from DCs (R&D Systems). VECTASHIELD® Antifade Mounting Medium (H-1000–10) and Anti-RAC1 (MBS9200589) were purchased from Vector Laboratories and MyBioSource, respectively, and used for confocal microscopy. 2-NBD-Glucose (186689-07-6) was used to measure glucose uptake (Abcam). DAPI (D9542–10MG), Tween® 20, viscous liquid, CAS 9005-64-5 (P1379) Ficoll-Paque PLUS (GE17-1440-03), Poly-L-lysine solution and Triton X-100 were acquired from Sigma-Aldrich. ActinRed 555 ReadyProbes Reagent and secondary antibodies Alexa Fluor 488 goat anti-rabbit IgG (H+L) and Alexa Fluor 488 goat anti-mouse IgG (H+L) were purchased from Invitrogen for confocal imaging. 10% Normal Goat Serum was also purchased for imaging (Life Technologies). For isolation of healthy patient dendritic cells, Blood Dendritic Cell Isolation Kit II, human (130-091-379) was purchased from Miltenyi Biotec.

Fluid shear stress application:

Six Brookfield cone-and-plate viscometers, which consist of a stationary plate in near contact with a rotating cone, were used to apply FSS to DCs throughout this study. The protocol used for these experiments followed that described previously.11,29 The advantage of using a cone-and-plate flow device for applying FSS is that the cells experience the same shear rate at all locations within the fluid volume.

A series of equations can thus be used to predict the local shear rate and stress. The equations are as follows:

G=ωtan(θ), where G is shear rate, ω is angular velocity (rad/s), and θ is the angle of the cone (rad).

τ = μG, where τ is the FSS and μ is the viscosity (cP). μ is expected to be 2.5 cP for these cell suspensions.

To verify that flow generated by the cone-and-plate device is laminar, the Reynold’s number can be calculated using the following equation:

Re#=r2a2ω12v, where r is radius, α is the angle of the cone, and v is kinematic viscosity. Re# is thus estimated to be 0.551. According to Buschmann, et al., flow in a cone-and-plate device is expected to be laminar with negligible secondary forces in applications with Reynold’s number up to the limiting number Re# = 1.293. Since the value for Re# in this experiment is below this critical value, flow can be assumed to be uniform across the plate.

Prior to flow experiments, surfaces of the cone-and-plate device were incubated with 5% bovine serum albumin (BSA) for 1 h to block nonspecific binding. In mouse serum albumin (MSA) control experiments, surfaces are blocked with 0.1% MSA. DC2.4 cells were lifted with trypsin, washed, and resuspended in complete RMPI media at 200,000 cells/mL. The viscometers were equipped with Brookfield Cone Part No. CPA-41Z spindles, accommodating a total of 2 mL of each sample. Cells were placed on a rotator to produce “static” conditions (<0.05 dyn/cm2). “0 dyn/cm2” was used as an additional control, where viscometers were set up identically to shear experiments, but the motors were left in an “off” state to ensure that nothing in the viscometer device was causing activation in the absence of force. For “shear” conditions, an FSS of 5 dyn/cm2 was applied for 1h using the cone-and-plate flow device unless otherwise indicated (Supplemental Figure 4). Static and shear conditions were either left untreated or treated with lipopolysaccharide (LPS), a potent stimulator, at 10 μg/mL.

Flow cytometry analysis and antibody staining:

Following FSS stimulation, cells were plated overnight or immediately prepared for flow cytometry analysis. For intracellular proteins such as phospho-NF-κB and -cFos, cells were fixed with 4% paraformaldehyde for 10 min. Cells were washed with HBSS and permeabilized with 100% ice cold methanol. Cells were washed again and stained for 15 min in the dark with antibodies fluorescently tagged with PE fluorophore while suspended in 1% BSA. A Guava easyCyte 12HT flow cytometer (Millipore, Billerica, MA) was used to measure fluorescence intensity, with FlowJo software used for gating and analysis. For extracellular proteins analyzed after 24 h, cells were lifted with trypsin, washed, and incubated with primary antibodies pre-conjugated with fluorophores in 1% BSA and then washed once more prior to analysis. For RAC1 staining, cells were first stained with the primary antibody and then stained for 15 min with an Alexa 488 secondary antibody and washed prior to analysis via flow cytometry

Glucose uptake analysis:

2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) is used as a fluorescent probe to analyze glucose uptake. 2-NBDG consists of a fluorescent d-glucose analog, which cells uptake in glucose-free media and is visualized using flow cytometry and confocal imaging.36 5 mg 2-NBDG was reconstituted in DMSO at a concentration of 25 mM. For this study, cells were exposed to 0 or 5 dyn/cm2 FSS for 1 h in glucose-free media, incubated with 35 μM 2-NBDG for 30 min in the dark, washed, and analyzed via flow cytometry.

pH measurements:

Immediately following applied FSS, the pH of the media was measured using a Thermo Scientific Orion Star A211 Benchtop Meter and pH Electrode Set. Calibrations were performed using 4.0, 7.0 and 10.0 pH buffers, and measurements were taken following calibration.

Annexin V assay:

Annexin V assay for apoptosis was performed to test the effects of FSS on cell viability, following previously established protocols.30,46–48 100,000 cells were collected following 1 h FSS at 5 dyn/cm2, washed, and resuspended in 150 μL HBSS with calcium and magnesium, and incubated with 3 μL annexin V (AV) and 3 μL propidium iodide (PI). Controls were prepared for unstained, AV only and PI only samples. Cells were incubated at RT for 15 min in the absence of light exposure. 100 μL of HBSS was added to each of the samples, which were then run through the flow cytometer. Cells that were negative for both AV and PI were identified as viable, as they lacked markers for apoptosis (AV) and necrosis (PI).

Cytokine staining:

For intracellular cytokine staining (ICS), cells were plated overnight following FSS stimulation. After 24 h, cells were treated with 1:1000 GolgiPlug Transport Inhibitor to block cytokine secretion. After 4 h, cells were lifted and stained following the previously described protocol for staining intracellular proteins. IL-6, IL-12 and CCR7 were investigated in this study. A two-way ANOVA test was used to determine statistical significance.

Proteome profiler:

DC2.4 cells were prepared as described above and brought to a final concentration of 1 × 106 cells/mL. FSS of 0 or 5 dyn/cm2 was applied to cells for 1 h and then cells were plated for 24–48 h to analyze cytokine expression using Proteome Profiler Mouse Cytokine Array Kit, Panel A (R&D Systems). The supernatant was collected from the cell suspensions and centrifuged at 4800 RPM for 10 min at 4°C. The manufacturer’s instructions were followed for the array procedure, and an Odyssey CLx imager was used for chemiluminescence imaging for 2 min exposure. Image Studio Lite software was used to quantify membrane fluorescence intensities.

Confocal microscopy:

Following the application of FSS, DCs were seeded onto glass coverslips previously coated with poly-l-lysine (PLL). After 4 h, cells were fixed with 4% paraformaldehyde and then permeabilized with 1% Triton. Cells were blocked with 5% BSA and 5% goat serum for 45 min, and later stained with DAPI nuclear stain and ActinRed 555 for 30 min. Coverslips were added to slides using Vectashield and cells were subsequently imaged using a Zeiss LSM 710 confocal microscope. Image analysis was performed using FIJI software. DCs with dendrite formation were defined as cells with 3 or more evident protrusions.

Isolation of bone marrow dendritic cells (BMDCs):

BMDCs were isolated following the protocol by Madaan, et al.49 In vivo studies were approved by Vanderbilt IACUC Protocol #M1700009–02. All methods using animals or animal-derived samples were carried out in accordance with relevant guidelines and regulations. Mice were monitored by staff from the Division of Animal Care (DAC) at Vanderbilt University. Femurs were harvested from healthy BALB/c mice and bone marrow was flushed out using HBSS and a 29G syringe. Bone marrow cells were treated with 20 ng/mL GM-CSF on Day 0, and on Day 3 10 mL fresh supplemented RPMI was added to the dish with 20 ng/mL GM-CSF. On Day 6, macrophages remined adhered and DCs were in suspension. DCs were collected and used for FSS experiments as previously described.

Human dendritic cell isolation:

Whole blood was collected from healthy human volunteers after informed consent. All experimental protocols involving humans or human tissue samples were approved by Vanderbilt University IRB Protocol #170222. All methods were carried out in accordance with relevant guidelines and regulations. Blood was collected in BD Vacutainer collection tubes with sodium citrate, with peripheral blood mononuclear cells (PBMCs) subsequently isolated using Ficoll-Paque gradient centrifugation. A heterogeneous mixture composed of plasmatycoid DCs (pDCs) and myeloid DCs (mDCs) were then isolated using Miltenyi Biotec’s Blood Dendritic Cell Isolation Kit II according to the manufacturer’s instructions. Cells were labeled and non-DCs were depleted, followed by labeling and positive selection of DCs. DCs resuspended in RPMI at 5 × 104 cells/mL and FSS was applied as described previously.

Statistics:

All data are reported as mean and standard error of the mean. Statistics were determined using student’s t test unless otherwise indicated. Each experiment included a minimum of three independent replicates unless otherwise indicated. Significance is indicated by *p<0.05, **p<0.01, ***p<0.005, and ****p<0.001. GraphPad Prism software was used to perform statistical comparisons and produce figures for this article.

Supplementary Material

1

ACKNOWLEDGEMENTS

Thank you to Dr. Rebecca Cook for assistance in preparing for the proteome profiler protocol.

FUNDING STATEMENT

This article was funded in part by National Institutes of Health, Grant No. CA256054 to M.R.K. and the NSF. This material is based on work supported by the National Science Foundation Graduate Research Fellowship Program to J.A.D Award Number 1937963.

Footnotes

CONFLICTS OF INTEREST

The authors have no conflicts of interest to disclose.

DECLARATION OF GENERATIVE AI AND AI-ASSISTED TECHNOLOGIES IN THE WRITING PROCESS

The authors did not use generative AI or AI-assisted technologies in the writing process.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

DATA AVAILABILITY STATEMENT

Raw data available on request to corresponding author mike.king@vanderbilt.edu.

REFERENCES

  • 1.Gu Y zhuo, Zhao X, Song X rong. Ex vivo pulsed dendritic cell vaccination against cancer. Acta Pharmacol Sin. 2020;41(7):959–969. doi: 10.1038/s41401-020-0415-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Crews DW, Dombroski JA, King MR. Prophylactic Cancer Vaccines Engineered to Elicit Specific Adaptive Immune Response. Front Oncol. 2021;11:626463. doi: 10.3389/fonc.2021.626463 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Huber A, Dammeijer F, Aerts JGJV, Vroman H Current State of Dendritic Cell-Based Immunotherapy: Opportunities for in vitro Antigen Loading of Different DC Subsets? Front Immunol. 2018;9:2804. doi: 10.3389/fimmu.2018.02804 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Anassi E, Ndefo UA. Sipuleucel-T (provenge) injection: the first immunotherapy agent (vaccine) for hormone-refractory prostate cancer. P T Peer-Rev J Formul Manag. 2011;36(4):197–202. [PMC free article] [PubMed] [Google Scholar]
  • 5.Cheever MA, Higano CS. PROVENGE (Sipuleucel-T) in Prostate Cancer: The First FDA-Approved Therapeutic Cancer Vaccine. Clin Cancer Res. 2011;17(11):3520–3526. doi: 10.1158/1078-0432.CCR-10-3126 [DOI] [PubMed] [Google Scholar]
  • 6.Zheng H, Chen J, Qiu W, et al. Safety and Efficacy of First-Line Treatments for Chemotherapy-Naive Metastatic Castration-Resistant Prostate Cancer: A Systematic Review and Indirect Comparison. BioMed Res Int. 2017;2017:1–10. doi: 10.1155/2017/3941217 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Fishman MN, Gupta, Carballido. Sipuleucel-T for therapy of asymptomatic or minimally symptomatic, castrate-refractory prostate cancer: an update and perspective among other treatments. OncoTargets Ther. Published online June 2011:79. doi: 10.2147/OTT.S14107 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.White CR, Frangos JA. The shear stress of it all: the cell membrane and mechanochemical transduction. Philos Trans R Soc B Biol Sci. 2007;362(1484):1459–1467. doi: 10.1098/rstb.2007.2128 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Nerem RM. Shear force and its effect on cell structure and function. ASGSB Bull Publ Am Soc Gravitational Space Biol. 1991;4(2):87–94. [PubMed] [Google Scholar]
  • 10.Yamamoto K, Korenaga R, Kamiya A, Ando J. Fluid Shear Stress Activates Ca2+ Influx Into Human Endothelial Cells via P2X4 Purinoceptors. Circ Res. 2000;87(5):385–391. doi: 10.1161/01.RES.87.5.385 [DOI] [PubMed] [Google Scholar]
  • 11.Hope JM, Lopez-Cavestany M, Wang W, Reinhart-King CA, King MR. Activation of Piezo1 sensitizes cells to TRAIL-mediated apoptosis through mitochondrial outer membrane permeability. Cell Death Dis. 2019;10(11):837. doi: 10.1038/s41419-019-2063-6 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Chistiakov DA, Orekhov AN, Bobryshev YV. Effects of shear stress on endothelial cells: go with the flow. Acta Physiol. 2017;219(2):382–408. doi: 10.1111/apha.12725 [DOI] [PubMed] [Google Scholar]
  • 13.Rossy J, Laufer JM, Legler DF. Role of Mechanotransduction and Tension in T Cell Function. Front Immunol. 2018;9:2638. doi: 10.3389/fimmu.2018.02638 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Haswell ES, Phillips R, Rees DC. Mechanosensitive channels: what can they do and how do they do it? Struct Lond Engl 1993. 2011;19(10):1356–1369. doi: 10.1016/j.str.2011.09.005 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Martinac B Mechanosensitive ion channels: molecules of mechanotransduction. J Cell Sci. 2004;117(12):2449–2460. doi: 10.1242/jcs.01232 [DOI] [PubMed] [Google Scholar]
  • 16.De Felice D, Alaimo A. Mechanosensitive Piezo Channels in Cancer: Focus on altered Calcium Signaling in Cancer Cells and in Tumor Progression. Cancers. 2020;12(7):1780. doi: 10.3390/cancers12071780 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Gerhold KA, Schwartz MA. Ion Channels in Endothelial Responses to Fluid Shear Stress. Physiology. 2016;31(5):359–369. doi: 10.1152/physiol.00007.2016 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Ando J, Yamamoto K. Flow detection and calcium signalling in vascular endothelial cells. Cardiovasc Res. 2013;99(2):260–268. doi: 10.1093/cvr/cvt084 [DOI] [PubMed] [Google Scholar]
  • 19.Swain SM, Liddle RA. Shear Stress-Induced Pathological Changes in Endothelial Cells Occur through Piezo1 Activation of TRPV4. Cell Biology; 2020. doi: 10.1101/2020.07.01.182212 [DOI] [Google Scholar]
  • 20.Sang L jie, Ju H qiang, Liu G ping, et al. LncRNA CamK-A Regulates Ca2+-Signaling-Mediated Tumor Microenvironment Remodeling. Mol Cell. 2018;72(1):71–83.e7. doi: 10.1016/j.molcel.2018.08.014 [DOI] [PubMed] [Google Scholar]
  • 21.Lee HJ, Diaz MF, Price KM, et al. Fluid shear stress activates YAP1 to promote cancer cell motility. Nat Commun. 2017;8(1):14122. doi: 10.1038/ncomms14122 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Moazzam F, DeLano FA, Zweifach BW, Schmid-Schonbein GW. The leukocyte response to fluid stress. Proc Natl Acad Sci. 1997;94(10):5338–5343. doi: 10.1073/pnas.94.10.5338 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Harrison DL, Fang Y, Huang J. T-Cell Mechanobiology: Force Sensation, Potentiation, and Translation. Front Phys. 2019;7:45. doi: 10.3389/fphy.2019.00045 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Liu CSC, Raychaudhuri D, Paul B, et al. Cutting Edge: Piezo1 Mechanosensors Optimize Human T Cell Activation. J Immunol. 2018;200(4):1255–1260. doi: 10.4049/jimmunol.1701118 [DOI] [PubMed] [Google Scholar]
  • 25.Hagihara M, Higuchi A, Tamura N, et al. Platelets, after Exposure to a High Shear Stress, Induce IL-10-Producing, Mature Dendritic Cells In Vitro. J Immunol. 2004;172(9):5297–5303. doi: 10.4049/jimmunol.172.9.5297 [DOI] [PubMed] [Google Scholar]
  • 26.Mitchell MJ, Lin KS, King MR. Fluid Shear Stress Increases Neutrophil Activation via Platelet-Activating Factor. Biophys J. 2014;106(10):2243–2253. doi: 10.1016/j.bpj.2014.04.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Shumilina E, Huber SM, Lang F. Ca2+ signaling in the regulation of dendritic cell functions. Am J Physiol-Cell Physiol. 2011;300(6):C1205–C1214. doi: 10.1152/ajpcell.00039.2011 [DOI] [PubMed] [Google Scholar]
  • 28.Lewis JS, Dolgova NV, Chancellor TJ, et al. The effect of cyclic mechanical strain on activation of dendritic cells cultured on adhesive substrates. Biomaterials. 2013;34(36):9063–9070. doi: 10.1016/j.biomaterials.2013.08.021 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Mitchell MJ, King MR. Fluid shear stress sensitizes cancer cells to receptor-mediated apoptosis via trimeric death receptors. New J Phys. 2013;15(1):015008. doi: 10.1088/1367-2630/15/1/015008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Mitchell MJ, Wayne E, Rana K, Schaffer CB, King MR. TRAIL-coated leukocytes that kill cancer cells in the circulation. Proc Natl Acad Sci. 2014;111(3):930–935. doi: 10.1073/pnas.1316312111 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Jyotsana N, Zhang Z, Himmel LE, Yu F, King MR. Minimal dosing of leukocyte targeting TRAIL decreases triple-negative breast cancer metastasis following tumor resection. Sci Adv. 2019;5(7):eaaw4197. doi: 10.1126/sciadv.aaw4197 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Fujii S ichiro, Liu K, Smith C, Bonito AJ, Steinman RM. The Linkage of Innate to Adaptive Immunity via Maturing Dendritic Cells In Vivo Requires CD40 Ligation in Addition to Antigen Presentation and CD80/86 Costimulation. J Exp Med. 2004;199(12):1607–1618. doi: 10.1084/jem.20040317 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Dilioglou S, Cruse JM, Lewis RE. Function of CD80 and CD86 on monocyte- and stem cell-derived dendritic cells. Exp Mol Pathol. 2003;75(3):217–227. doi: 10.1016/S0014-4800(03)00072-8 [DOI] [PubMed] [Google Scholar]
  • 34.Sprent J Antigen-Presenting Cells: Professionals and amateurs. Curr Biol. 1995;5(10):1095–1097. doi: 10.1016/S0960-9822(95)00219-3 [DOI] [PubMed] [Google Scholar]
  • 35.Blanco P, Palucka A, Pascual V, Banchereau J. Dendritic cells and cytokines in human inflammatory and autoimmune diseases. Cytokine Growth Factor Rev. 2008;19(1):41–52. doi: 10.1016/j.cytogfr.2007.10.004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Zou C, Wang Y, Shen Z. 2-NBDG as a fluorescent indicator for direct glucose uptake measurement. J Biochem Biophys Methods. 2005;64(3):207–215. doi: 10.1016/j.jbbm.2005.08.001 [DOI] [PubMed] [Google Scholar]
  • 37.Benvenuti F, Hugues S, Walmsley M, et al. Requirement of Rac1 and Rac2 Expression by Mature Dendritic Cells for T Cell Priming. Science. 2004;305(5687):1150–1153. doi: 10.1126/science.1099159 [DOI] [PubMed] [Google Scholar]
  • 38.Liu T, Zhang L, Joo D, Sun SC. NF-κB signaling in inflammation. Signal Transduct Target Ther. 2017;2(1):17023. doi: 10.1038/sigtrans.2017.23 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Vuong B, Hogan-Cann ADJ, Alano CC, et al. NF-κB transcriptional activation by TNFα requires phospholipase C, extracellular signal-regulated kinase 2 and poly(ADP-ribose) polymerase-1. J Neuroinflammation. 2015;12(1):229. doi: 10.1186/s12974-015-0448-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Ouaaz F, Arron J, Zheng Y, Choi Y, Beg AA. Dendritic Cell Development and Survival Require Distinct NF-κB Subunits. Immunity. 2002;16(2):257–270. doi: 10.1016/S1074-7613(02)00272-8 [DOI] [PubMed] [Google Scholar]
  • 41.Rescigno M, Martino M, Sutherland CL, Gold MR, Ricciardi-Castagnoli P. Dendritic Cell Survival and Maturation Are Regulated by Different Signaling Pathways. J Exp Med. 1998;188(11):2175–2180. doi: 10.1084/jem.188.11.2175 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Ade N, Antonios D, Kerdine-Romer S, Boisleve F, Rousset F, Pallardy M. NF-κB Plays a Major Role in the Maturation of Human Dendritic Cells Induced by NiSO4 but not by DNCB. Toxicol Sci. 2007;99(2):488–501. doi: 10.1093/toxsci/kfm178 [DOI] [PubMed] [Google Scholar]
  • 43.Shaulian E, Karin M. AP-1 in cell proliferation and survival. Oncogene. 2001;20(19):2390–2400. doi: 10.1038/sj.onc.1204383 [DOI] [PubMed] [Google Scholar]
  • 44.Atsaves V, Leventaki V, Rassidakis GZ, Claret FX. AP-1 Transcription Factors as Regulators of Immune Responses in Cancer. Cancers. 2019;11(7):E1037. doi: 10.3390/cancers11071037 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Westdorp H, Sköld AE, Snijer BA, et al. Immunotherapy for Prostate Cancer: Lessons from Responses to Tumor-Associated Antigens. Front Immunol. 2014;5. doi: 10.3389/fimmu.2014.00191 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Hope JM, Bersi MR, Dombroski JA, et al. Circulating prostate cancer cells have differential resistance to fluid shear stress-induced cell death. J Cell Sci. 2021;134(4):jcs251470. doi: 10.1242/jcs.251470 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Dombroski JA, Jyotsana N, Crews DW, Zhang Z, King MR. Fabrication and Characterization of Tumor Nano-Lysate as a Preventative Vaccine for Breast Cancer. Langmuir. 2020;36(23):6531–6539. doi: 10.1021/acs.langmuir.0c00947 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Grayson KA, Hope JM, Wang W, Reinhart-King CA, King MR. Taxanes Sensitize Prostate Cancer Cells to TRAIL-Induced Apoptotic Synergy via Endoplasmic Reticulum Stress. Mol Cancer Ther. 2021;20(5):833–845. doi: 10.1158/1535-7163.MCT-20-0495 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Madaan A, Verma R, Singh AT, Jain SK, Jaggi M. A stepwise procedure for isolation of murine bone marrow and generation of dendritic cells. J Biol Methods. 2014;1(1):e1. doi: 10.14440/jbm.2014.12 [DOI] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1933132-supplement-1.pdf (548.8KB, pdf)

Data Availability Statement

Raw data available on request to corresponding author mike.king@vanderbilt.edu.

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