Long-term expression changes of immune-related genes in prostate cancer after radiotherapy.

The expression of immune-related genes in cancer cells can alter the anti-tumor immune response and thereby impact patient outcomes. Radiotherapy has been shown to modulate immune-related genes dependent on the fractionation regimen. To identify long-term changes in gene expression after irradiation, PC3 (p53 deleted) and LNCaP (p53 wildtype) prostate cancer cells were irradiated with either a single dose (SD, 10 Gy) or a fractionated regimen (MF) of 10 fractions (1 Gy per fraction). Whole human genome arrays were used to determine gene expression at 24 h and 2 months after irradiation. Immune pathway activation was analyzed with Ingenuity Pathway Analysis software. Additionally, 3D colony formation assays and T-cell cytotoxicity assays were performed. LNCaP had a higher basal expression of immunogenic genes and was more efficiently killed by cytotoxic T-cells compared to PC3. In both cell lines, MF irradiation resulted in an increase in multiple immune-related genes immediately after irradiation, while at 2 months, SD irradiation had a more pronounced effect on radiation-induced gene expression. Both immunogenic and immunosuppressive genes were upregulated in the long term in PC3 cells by a 10 Gy SD irradiation but not in LNCaP. T-cell-mediated cytotoxicity was significantly increased in 10 Gy SD PC3 cells compared to the unirradiated control and could be further enhanced by treatment with immune checkpoint inhibitors. Irradiation impacts the expression of immune-related genes in cancer cells in a fractionation-dependent manner. Understanding and targeting these changes may be a promising strategy for primary prostate cancer and recurrent tumors.

C3aR Signaling Inhibits NK-cell Infiltration into the Tumor Microenvironment in Mouse Models.

Many solid tumors have low levels of cytotoxic CD56dim natural killer (NK) cells, suggesting that CD56dim NK-cell exclusion from the tumor microenvironment (TME) contributes to the decreased response rate of immunotherapy. Complement component 3a (C3a) is known for its tumor-promoting and immunosuppressive roles in solid tumors. Previous reports have implicated the involvement of the C3a receptor (C3aR) in immune cell trafficking into the TME. C3aR is predominantly expressed on the surface of activated cytotoxic NK cells, but a specific role for C3aR in NK-cell biology has not been investigated. Because solid tumors generate elevated C3a and have decreased NK-cell infiltration, we hypothesized that C3aR might play a role in cytotoxic NK-cell recruitment into the TME. Our results indicate that blocking C3aR signaling in NK cells increased NK-cell infiltration into the TME in mouse models and led to tumor regression. Because the critical lymphocyte trafficking integrin LFA-1 orchestrates the migration of activated NK cells, we wanted to gain insight into the interaction between C3aR signaling and LFA-1. Our results demonstrated that direct interaction between C3aR and LFA-1, which led to a high-affinity LFA-1 conformation, decreased NK-cell infiltration into the TME. We propose that approaches to enhance cytotoxic NK-cell infiltration into the TME, through either disrupting C3a and C3aR interaction or inhibiting the formation of high-affinity LFA-1, represent a new strategy to improve the efficiency of immunotherapy for cancer treatment.

Neutralization of PD-L2 is Essential for Overcoming Immune Checkpoint Blockade Resistance in Ovarian Cancer.

PURPOSE: Ovarian cancer represents a major clinical hurdle for immune checkpoint blockade (ICB), with reported low patient response rates. We found that the immune checkpoint ligand PD-L2 is robustly expressed in patient samples of ovarian cancers and other malignancies exhibiting suboptimal response to ICB but not in cancers that are ICB sensitive. Therefore, we hypothesize that PD-L2 can facilitate immune escape from ICB through incomplete blockade of the PD-1 signaling pathway. EXPERIMENTAL DESIGN: We engineered a soluble form of the PD-1 receptor (sPD-1) capable of binding and neutralizing both PD-L2 and PD-L1 with ×200 and ×10,000 folds improvement in binding affinity over wild-type PD-1 leading to superior inhibition of ligand-mediated PD-1 activities. RESULTS: Both in vitro and in vivo analyses performed in this study demonstrated that the high-affinity sPD-1 molecule is superior at blocking both PD-L1- and PD-L2-mediated immune evasion and reducing tumor growth in immune-competent murine models of ovarian cancer. CONCLUSIONS: The data presented in this study provide justification for using a dual targeting, high-affinity sPD-1 receptor as an alternative to PD-1 or PD-L1 therapeutic antibodies for achieving superior therapeutic efficacy in cancers expressing both PD-L2 and PD-L1.

Chemorepellent Semaphorin 3E Negatively Regulates Neutrophil Migration In Vitro and In Vivo.

Neutrophil migration is an essential step in leukocyte trafficking during inflammatory responses. Semaphorins, originally discovered as axon guidance cues in neural development, have been shown to regulate cell migration beyond the nervous system. However, the potential contribution of semaphorins in the regulation of neutrophil migration is not well understood. This study examines the possible role of a secreted chemorepellent, Semaphorin 3E (Sema3E), in neutrophil migration. In this study, we demonstrated that human neutrophils constitutively express Sema3E high-affinity receptor, PlexinD1. Sema3E displayed a potent ability to inhibit CXCL8/IL-8-induced neutrophil migration as determined using a microfluidic device coupled to real-time microscopy and a transwell system in vitro. The antimigratory effect of Sema3E on human neutrophil migration was associated with suppression of CXCL8/IL-8-mediated Ras-related C3 botulinum toxin substrate 1 GTPase activity and actin polymerization. We further addressed the regulatory role of Sema3E in the regulation of neutrophil migration in vivo. Allergen airway exposure induced higher neutrophil recruitment into the lungs of Sema3e-/- mice compared with wild-type controls. Administration of exogenous recombinant Sema3E markedly reduced allergen-induced neutrophil recruitment into the lungs, which was associated with alleviation of allergic airway inflammation and improvement of lung function. Our data suggest that Sema3E could be considered an essential regulatory mediator involved in modulation of neutrophil migration throughout the course of neutrophilic inflammation.

Microfluidic-Based Live-Cell Analysis of NK Cell Migration In Vitro.

Effector functions and cellular properties of natural killer (NK) cells are regulated by cellular and extracellular factors shaped by the microenvironments. NK cells express specific chemokine and non-chemokine receptors to aid preferential migrations or localizations in tissues. Good understanding of how NK-cell migratory properties are regulated in physiological and pathological microenvironments will provide further insights into the development of NK cell-based therapeutic approaches. In contrast to the commonly used conventional in vitro migration assays such as Trans-well assays that measure movements of a population of the migratory cells, microfluidic-based devices support live-cell imaging of cell migrations under a well-defined chemical gradient(s) at microscale. Subsequent analyses at single-cell level provide quantitative measurements of cell-migration parameters such as speed and Chemotactic Index, and permit distinguishing chemotaxis, chemokinesis, and chemo-repulsion. Our recent work established the use of a Y-shaped microfluidic device to study NK cell migrations in vitro. In this chapter, we described the detailed method of acquiring and analyzing NK cell migration in the microfluidic devices.

Bidirectional interactions of NK cells and dendritic cells in immunotherapy: current and future perspective.

NK cells and dendritic cells (DC) are innate cellular components that regulate adaptive immune responses in the immune surveillance of cancer and infections. Interactions of NK and DC are bidirectional. In this mini review, we summarized how NK cells regulate immature DC editing and maturation, how DC regulate NK-cell functions reciprocally in the NK-DC crosstalk, and the importance of NK-DC crosstalk in antitumor immunity. Enhancing NK-DC crosstalk by cellular factor(s), antibodies or creating a microenvironment that promote NK activations, DC maturation and NK-DC crosstalk will provide new insights into future development of DC-based immunotherapy.

Microfluidic-based, live-cell analysis allows assessment of NK-cell migration in response to crosstalk with dendritic cells.

Migration and localization of NK cells into peripheral tissues are tightly regulated under normal and pathological conditions. The physiological importance of NK cell-DC crosstalk has been well documented. However, the ways in which DCs regulate the migratory properties of NK cells (such as chemotaxis, chemokinesis, chemo-repulsion) are not fully defined in vitro. Here, we employed a microfluidic platform to examine, at the single-cell level, C57BL/6 NK-cell migrations in a stable chemical gradient. We observed that soluble factors released by the immature and LPS-activated mature DCs induced a high level of chemotactic movement of IL-2-activated NK cells in vitro. We confirmed these findings in a standard trans-well migration assay, and identified CXCR3 as a key receptor on the NK cells that mediated the migration. More interestingly, we revealed a novel function of granulocyte macrophage colony-stimulating factor in repulsing NK-cell migrations. The future uses of such microfluidic device in the systematic evaluations of NK-cell migratory responses in NK cell-DC crosstalk will provide new insights into the development of DC-based NK-cell therapies against tumor and infections.

Effects of Clostridium difficile toxin A and B on human T lymphocyte migration.

Bacterial products such as toxins can interfere with a variety of cellular processes, leading to severe human diseases. Clostridium difficile toxins, TcdA and TcdB are the primary contributing factors to the pathogenesis of C. difficile-associated diseases (CDAD). While the mechanisms for TcdA and TcdB mediated cellular responses are complex, it has been shown that these toxins can alter chemotactic responses of neutrophils and intestinal epithelial cells leading to innate immune responses and tissue damages. The effects of C. difficile toxins on the migration and trafficking of other leukocyte subsets, such as T lymphocytes, are not clear and may have potential implications for adaptive immunity. We investigated here the direct and indirect effects of TcdA and TcdB on the migration of human blood T cells using conventional cell migration assays and microfluidic devices. It has been found that, although both toxins decrease T cell motility, only TcdA but not TcdB decreases T cell chemotaxis. Similar effects are observed in T cell migration toward the TcdA- or TcdB-treated human epithelial cells. Our study demonstrated the primary role of TcdA (compared to TcdB) in altering T cell migration and chemotaxis, suggesting possible implications for C. difficile toxin mediated adaptive immune responses in CDAD.

The tandem PH domain-containing protein 2 (TAPP2) regulates chemokine-induced cytoskeletal reorganization and malignant B cell migration.

The intracellular signaling processes controlling malignant B cell migration and tissue localization remain largely undefined. Tandem PH domain-containing proteins TAPP1 and TAPP2 are adaptor proteins that specifically bind to phosphatidylinositol-3,4-bisphosphate, or PI(3,4)P2, a product of phosphoinositide 3-kinases (PI3K). While PI3K enzymes have a number of functions in cell biology, including cell migration, the functions of PI(3,4)P2 and its binding proteins are not well understood. Previously we found that TAPP2 is highly expressed in primary leukemic B cells that have strong migratory capacity. Here we find that SDF-1-dependent migration of human malignant B cells requires both PI3K signaling and TAPP2. Migration in a transwell assay is significantly impaired by pan-PI3K and isoform-selective PI3K inhibitors, or by TAPP2 shRNA knockdown (KD). Strikingly, TAPP2 KD in combination with PI3K inhibitor treatment nearly abolished the migration response, suggesting that TAPP2 may contribute some functions independent of the PI3K pathway. In microfluidic chamber cell tracking assays, TAPP2 KD cells show reduction in percentage of migrating cells, migration velocity and directionality. TAPP2 KD led to alterations in chemokine-induced rearrangement of the actin cytoskeleton and failure to form polarized morphology. TAPP2 co-localized with the stable F-actin-binding protein utrophin, with both molecules reciprocally localizing against F-actin accumulated at the leading edge upon SDF-1 stimulation. In TAPP2 KD cells, Rac was over-activated and localized to multiple membrane protrusions, suggesting that TAPP2 may act in concert with utrophin and stable F-actin to spatially restrict Rac activation and reduce formation of multiple membrane protrusions. TAPP2 function in cell migration is also apparent in the more complex context of B cell migration into stromal cell layers - a process that is only partially dependent on PI3K and SDF-1. In summary, this study identified TAPP2 as a novel regulator of malignant B cell migration and a potential therapeutic intervention target.

Growth and positioning of adipose-derived stem cells in microfluidic devices.

Stem cells hold great promise for treatment of various degenerative diseases. However, clinical studies have only shown very moderate benefits of cell therapy. We believe that insufficiency of therapeutic benefits is due to limited homing of implanted stem cells to targeted organs. Microfluidic devices are a very useful research tool for quantitative characterizations of stem cells. The present study therefore was to assess the effects of epidermal growth factor (EGF) and direct current electric field (dcEF) on the growth and trafficking of adipose-derived stem cells (ASC). It was found that EGF did not affect cell proliferation in cell-culture flasks. However, ASC proliferated at a higher rate in microfluidic devices with continuous infusion of EGF. Furthermore, we found that ASC migrated toward an EGF gradient in microfluidic devices. Moreover, we found that ASC tended to position perpendicularly to dcEF. The results suggest that EGF and dcEF may be effective in guiding homing and trafficking of implanted ASC.

Combinatorial guidance by CCR7 ligands for T lymphocytes migration in co-existing chemokine fields.

Chemokines mediate the trafficking and positioning of lymphocytes in lymphoid tissues that is crucial for immune surveillance and immune responses. In particular, a CCR7 ligand, CCL21, plays important roles in recruiting T cells to secondary lymphoid tissues (SLT). Furthermore, CCL21 together with another CCR7 ligand, CCL19, direct the navigation and compartmentation of T cells within SLT. However, the distinct roles of these two chemokines for regulating cell trafficking and positioning are not clear. In this study, we explore the effect of co-existing CCL19 and CCL21 concentration fields on guiding T cell migration. Using microfluidic devices that can configure single and superimposed chemokine fields we show that under physiological gradient conditions, human peripheral blood T cells chemotax to CCL21 but not CCL19. Furthermore, T cells migrate away from the CCL19 gradient in a uniform background of CCL21. This repulsive migratory response is predicted by mathematical modeling based on the competition of CCL19 and CCL21 for CCR7 signaling and the differential ability of the two chemokines for desensitizing CCR7. These results suggest a new combinatorial guiding mechanism by CCL19 and CCL21 for the migration and trafficking of CCR7 expressing leukocytes.

Microfluidics for food, agriculture and biosystems industries.

Microfluidics, a rapidly emerging enabling technology has the potential to revolutionize food, agriculture and biosystems industries. Examples of potential applications of microfluidics in food industry include nano-particle encapsulation of fish oil, monitoring pathogens and toxins in food and water supplies, micro-nano-filtration for improving food quality, detection of antibiotics in dairy food products, and generation of novel food structures. In addition, microfluidics enables applications in agriculture and animal sciences such as nutrients monitoring and plant cells sorting for improving crop quality and production, effective delivery of biopesticides, simplified in vitro fertilization for animal breeding, animal health monitoring, vaccination and therapeutics. Lastly, microfluidics provides new approaches for bioenergy research. This paper synthesizes information of selected microfluidics-based applications for food, agriculture and biosystems industries.

Activated T lymphocytes migrate toward the cathode of DC electric fields in microfluidic devices.

Immune cell migration is a fundamental process that enables immunosurveillance and immune responses. Understanding the mechanism of immune cell migration is not only of importance to the biology of cells, but also has high relevance to cell trafficking mediated physiological processes and diseases such as embryogenesis, wound healing, autoimmune diseases and cancers. In addition to the well-known chemical concentration gradient based guiding mechanism (i.e. chemotaxis), recent studies have shown that lymphocytes can respond to applied physiologically relevant direct current (DC) electric fields by migrating toward the cathode of the fields (i.e. electrotaxis) in both in vitro and in vivo settings. In the present study, we employed two microfluidic devices allowing controlled application of electric fields inside the microfluidic channel for quantitative studies of lymphocyte electrotaxis in vitro at the single cell level. The first device is fabricated by soft-lithography and the second device is made in glass with integrated on-chip electrodes. Using both devices, we for the first time showed that anti-CD3/CD28 antibodies activated human blood T cells migrate to the cathode of the applied DC electric field. This finding is consistent with previous electrotaxis studies on other lymphocyte subsets suggesting electrotaxis is a novel guiding mechanism for immune cell migration. Furthermore, the characteristics of electrotaxis and chemotaxis of activated T cells in PDMS microfluidic devices are compared.