Acousto-optofluidic 3D single cell imaging of macrophage phagocytosis of Pseudomonas Aeruginosa.

Understanding how immune cells such as monocytes or macrophages within our blood and tissue engulf and destroy foreign organisms is important for developing new therapies. The process undertaken by these cells, called phagocytosis, has yet to be observed in real-time at the single cell level. Microfluidic-based imaging platforms offer a wide range of tools for precise fluid control and biomolecule manipulation that makes regulating long term experiments and data collection possible. With the compatibility between acoustofluidics and light-sheet fluorescent microscopy (LSFM) previously demonstrated, here an acousto-optfluidic device with on-chip fluid flow direction control was developed. The standing surface acoustic waves (SSAWs) were used to trap, load and safeguard individual cells within a highly controllable fluid loop, created via the triggering of on-chip PDMS valves, to demonstrate multiple rounds of live single cell imaging. The valves allowed for the direction of the fluid flow to be changed (between forward and reverse operation) without altering the inlet flow rate, an important factor for performing reproducible and comparable imaging of samples over time. With this high-resolution imaging system, volumetric reconstructions of phagocytosed bacteria within macrophages could be resolved over a total of 9 rounds of imaging: totalling 19 reconstructed images of the cell membrane with visible intracellular bacteria.

Student Performance on an Objective Structured Clinical Exam Delivered Both Virtually and In-Person.

OBJECTIVE: Passing a milestone objective structured clinical examination (OSCE) is a graduation requirement for the University of Waterloo Pharmacy students. In January 2021, the milestone OSCE was offered concurrently both virtually and in-person, with students being able to choose their desired format. The purpose of this study was to compare student performance between the 2 formats and to identify factors that may have predicted student choice of format. METHODS: Objective structured clinical examination scores for in-person and virtual exam-takers were compared using 2-tailed independent t tests with Bonferroni correction. Pass rates were compared using χ2 analysis. Prior academic performance variables were analyzed to identify predictors of the chosen exam format. Student and exam personnel surveys were used to capture OSCE feedback. RESULTS: A total of 67 students (56%) participated in the in-person OSCE, and 52 students (44%) participated virtually. There were no significant differences in overall exam averages or pass rates between the 2 groups. However, virtual exam-takers scored lower in 2 of 7 cases. Previous academic performance did not predict the choice of exam format. Feedback surveys indicated that the exam organization was perceived as a strength regardless of format, but in-person students felt more prepared for the exam than virtual exam-takers with technical challenges and difficulty navigating station resources being noted as barriers in the virtual offering. CONCLUSION: Virtual and in-person administration of a milestone OSCE resulted in similar student performance, with slightly lower performance on 2 individual case scores with virtual delivery. These results may inform the future development of virtual OSCEs.

A longitudinal qualitative study examining the impact of Check-In, a novel learning activity on burnout for pharmacy students.

INTRODUCTION: Burnout is a phenomenon that can occur in any occupation, but pharmacists may be more prone to its effects. Because of its relevance to the pharmacy profession, the University of Waterloo School of Pharmacy incorporated an active learning activity to teach pharmacy students about healthcare provider burnout. This activity, named Check-In, was launched in March 2020 and consisted of one-on-one check-ins between pharmacy students and staff. As the first learning activity of its kind, the researchers wished to investigate the ongoing impact of Check-In. METHODS: This qualitative, descriptive study was composed of telephone interviews with 13 students that partook in Check-In. These interviews were held at two different timepoints: six and 18 months post-activity. Participants were recruited until data saturation, and transcripts underwent thematic analysis. RESULTS: Four themes were identified from the interviews: (1) Check-In was a valuable learning activity; (2) students performed some form of checking in post-activity; (3) burnout could be recognized and defined by students; (4) Check-In's place in the pharmacy curriculum still needs to be determined. CONCLUSIONS: Check-In is an innovative learning activity to teach pharmacy learners about healthcare provider burnout. It can be utilized by pharmacy institutions to incorporate wellness and student mental health into curriculum.

Acoustofluidic cell micro-dispenser for single cell trajectory control.

The precise manipulation of individual cells is a key capability for the study of single cell physiological characteristics or responses to stimuli. Currently, only large cell populations can be transferred with certainty using expensive and laborious flow cytometry platforms. However, when approaching small populations of cells, this task becomes increasingly challenging. Here, we report an effective acoustofluidic micro-dispenser, utilising surface acoustic waves (SAWs), with the ability to trap and release cells on demand, which when combined with an external valve can guide the trajectory of individual cells. We demonstrate single cell trap and release with a single cell trapping effectiveness of 74%, enabling the capability of dispensing a highly controlled amount of cells without any harmful effects. This device has the potential to be easily integrated into a wide range of analytical platforms for applications such as single cell fluorescent imaging and single cell proteomic studies.

Check-In: An Educational Activity to Address Well-Being and Burnout among Pharmacy Students.

Background: Chronic workplace stress that has not been adequately managed can result in burnout. Healthcare providers; including pharmacists, may be particularly susceptible to this phenomenon, prompting the School of Pharmacy at the University of Waterloo to develop an active-learning activity to teach and reflect on healthcare provider burnout, called Check-In. Methods: Check-In was comprised of a 20 min online lecture on healthcare provider burnout, two pre-readings that highlighted burnout among physicians, and an optional one-on-one session between individual students and a faculty or staff member. A reflection guide was also shared among students and facilitators where students had to rate their current mental health on a 10-point scale and reflect on questions focusing on energy expenditure, self-care, and self-compassion within the past, present, and future. Results: Check-In was rewarding and overall positive for students and faculty. The personal connection with members from the school and the strategic timing of the activity within the curriculum notably contributed to the success of the activity. The short duration of individual sessions was the key criticism of the activity. Further research at the University of Waterloo School of Pharmacy will be explored to assess the long-term impact of Check-In on student well-being.

The Design and Impact of an Interprofessional Education Event Among Pharmacy and Nursing Students in Palliative Care-RnRx.

Nearly all reports of interprofessional education (IPE) in palliative care have excluded pharmacy students. This article describes an IPE event between pharmacy and nursing students and assesses its impact on IPE competencies. Second-year nursing students and third-year pharmacy students participated in an evening-long event, focused on a married couple who each require palliative care-one for end-of-life planning and one for chronic disease progression. The impact of the event was assessed using the Interprofessional Collaborative Competency Attainment Scale (ICCAS) and qualitative feedback. Two hundred nine (96.7%) completed the ICCAS, and 16 of the 20 statements of the ICCAS showed large positive effect sizes (Cohen d ≥ 0.8), with the remaining 4 showing moderate positive effect sizes (Cohen d ≥ 0.5). The greatest effect sizes were related to improved awareness of complementary skillsets and knowledge between the professions. Addressing team conflict and including the patient/family in decision-making showed the least improvement. While ongoing interactions are ideal for the development of skills related to conflict and team development, this article demonstrates that even a 1-time activity can have an impact on students' interprofessional care competence.

The emerging role of microfluidics in multi-material 3D bioprinting.

To assist the transition of 3D bioprinting technology from simple lab-based tissue fabrication, to fully functional and implantable organs, the technology must not only provide shape control, but also functional control. This can be accomplished by replicating the cellular composition of the native tissue at the microscale, such that cell types interact to provide the desired function. There is therefore a need for precise, controllable, multi-material printing that could allow for high, possibly even single cell, resolution. This paper aims to draw attention to technological advancements made in 3D bioprinting that target the lack of multi-material, and/or multi cell-type, printing capabilities of most current devices. Unlike other reviews in the field, which largely focus on variations in single-material 3D bioprinting involving the standard methods of extrusion-based, droplet-based, laser-based, or stereolithographic methods; this review concentrates on sophisticated multi-material 3D bioprinting using multi-cartridge printheads, co-axial nozzles and microfluidic-enhanced printing nozzles.

Partners in pharmacy: An intraprofessional educational event with pharmacy and pharmacy technician students.

BACKGROUND AND PURPOSE: Upon graduation and licensing, pharmacists work very closely with pharmacy technicians. Despite this, opportunities for learning together as students are limited. We developed and implemented a pilot intraprofessional event for pharmacy and pharmacy technician students. The purpose of this study was to evaluate the perceived value and learner confidence through analysis of participant feedback. EDUCATION ACTIVITY AND SETTING: Pharmacy students from the University of Waterloo School of Pharmacy and pharmacy technician students from Lambton College participated in an intraprofessional event that included a three-station practice objective structured clinical exam (OSCE) and a case discussion regarding a methadone dispensing error, followed by a facilitated debrief. Upon completion of the event, students were invited to complete an online feedback questionnaire. FINDINGS: Twenty-one pharmacy students and 22 pharmacy technician students participated in the event. Twenty-one students completed the questionnaire, for a response rate of 49%. The majority of respondents agreed or strongly agreed that the event enhanced learning and confidence in working together to provide interprofessional care. Students seemed to find the OSCE to be particularly valuable. Feedback suggestions for improvement indicated a desire for more activities and time allocated to the event. SUMMARY: We designed and implemented a pilot intraprofessional event that was well-received by pharmacy students and pharmacy technician students. This supports the development of future similar events.

Blood platelet enrichment in mass-producible surface acoustic wave (SAW) driven microfluidic chips.

The ability to separate specific biological components from cell suspensions is indispensable for liquid biopsies, and for personalized diagnostics and therapy. This paper describes an advanced surface acoustic wave (SAW) based device designed for the enrichment of platelets (PLTs) from a dispersion of PLTs and red blood cells (RBCs) at whole blood concentrations, opening new possibilities for diverse applications involving cell manipulation with high throughput. The device is made of patterned SU-8 photoresist that is lithographically defined on the wafer scale with a new proposed methodology. The blood cells are initially focused and subsequently separated by an acoustic radiation force (ARF) applied through standing SAWs (SSAWs). By means of flow cytometric analysis, the PLT concentration factor was found to be 7.7, and it was proven that the PLTs maintain their initial state. A substantially higher cell throughput and considerably lower applied powers than comparable devices from literature were achieved. In addition, fully coupled 3D numerical simulations based on SAW wave field measurements were carried out to anticipate the coupling of the wave field into the fluid, and to obtain the resulting pressure field. A comparison to the acoustically simpler case of PDMS channel walls is given. The simulated results show an ideal match to the experimental observations and offer the first insights into the acoustic behavior of SU-8 as channel wall material. The proposed device is compatible with current (Lab-on-a-Chip) microfabrication techniques allowing for mass-scale, reproducible chip manufacturing which is crucial to push the technology from lab-based to real-world applications.

Impact and Attitudes about Peer Review of Teaching in a Canadian Pharmacy School.

Objective. To evaluate attitudes toward peer review of teaching and its impact on teaching practices and perceptions. Methods. The University of Waterloo School of Pharmacy implemented a peer-review process for its teaching program in 2015. Those reviewed were invited to complete an electronic survey that captured their attitudes toward teaching, attitudes toward peer review, and changes in teaching practices, and to participate in semi-structured follow-up interviews for more in-depth discussion of these issues. Results. Twenty-six (76%) instructors completed the survey. Instructors agreed that peer reviews of teaching are a development opportunity (96%), and 73% were comfortable with the idea of peer review. Over half (58%) indicated that the review made them feel more confident that their teaching strategies were effective, and the same percentage indicated that they planned to make changes to their teaching as a result of the feedback received from the peer review. Only a few instructors indicated that peer review changed their attitudes toward teaching (12%) or increased the value they placed on teaching (34%). Eight instructors (23.5%) participated in the semi-structured interviews. Themes that emerged included: attempts to make the reviewee comfortable during the peer review were successful; the feedback provided to instructors regarding their teaching was positive but not critical enough; there was lack of clarity as to the purpose of the feedback; and instructors planned to make only minor changes to their teaching as a result of the review. Conclusion. Peer review of teaching was well received and feedback was confirmatory in nature but had minimal impact on teaching practices as it was not deemed to be critical enough. Changes to the peer review program are needed to increase its impact on teaching practices.

Addressing professionalism in both formative and summative simulated patient care activities - Experiences with a discretionary deduction approach.

BACKGROUND AND PURPOSE: Encouraging and assessing professionalism among pharmacy students can be challenging. While non-punitive approaches are preferred as part of professional socialization, our program found this insufficient to ensure professional behaviour, especially during ungraded simulation lab activities. EDUCATIONAL ACTIVITY AND SETTING: In Winter 2015, we included a discretionary grade deduction within the assessments applied to a professional practice lab course in order to provide both intrinsic and extrinsic motivation to follow lab policies on professionalism. FINDINGS: A professionalism code was developed and discussed with students, and was also used as a template to guide professional behaviour throughout the course. Students not exhibiting these behaviours in lab could be subject to up to a five percent deduction from their final course grade at the instructor's discretion. DISCUSSION: Instructors considering this strategy are encouraged to introduce it in the first year of the program to ensure consistent expectations throughout the duration of students' training, and to not determine the magnitude of deductions applied until the end of the semester to ensure consistency and consideration of ongoing behaviour. Documentation of actions leading to deductions should be kept to support the decision. SUMMARY: A deduction-only approach to address unprofessional behaviours in addition to discussion offers additional motivation to students to exhibit professionalism across both graded and ungraded educational activities with minimal additional workload for instructors. The strategy has since been adopted across all lab courses in our program and has also been recommended in lecture-based courses.

Evaluation of a Unique Interprofessional Education Program Involving Medical and Pharmacy Students.

Objective. To measure changes in interprofessional competencies among pharmacy and medical students following a half-day event focusing on interprofessional learning. Methods. There were 118 pharmacy students and 28 medical students who participated in the Healthcare Interprofessional Education Day (HIPED) which consisted of three stations (communication, patient interviewing, and prescribing) in which pharmacy and medical students had to work collaboratively. The standardized Interprofessional Collaborative Competency Attainment Survey (ICCAS) was used to evaluate the effectiveness of the program. Results. There were 133 surveys completed for a response rate of 91%. All 20 items measured by the ICCAS showed a significant improvement. The strongest effect sizes were in the collaboration, roles & responsibilities, and collaborative practice/family-centered approach categories. The least robust effects were in the conflict management/resolution category. Conclusion. The HIPED activity was an effective IPE experience. The strong and consistent improvement in all ICCAS scores suggest a framework for pharmacy and medical school training to move from siloed educational experiences to synergistic learning opportunities.

Chemotherapeutic agents attenuate CXCL12-mediated migration of colon cancer cells by selecting for CXCR4-negative cells and increasing peptidase CD26.

BACKGROUND: Recurrence of colorectal cancer (CRC) may arise due to the persistence of drug-resistant and cancer-initiating cells that survive exposure to chemotherapy. Proteins responsible for this recurrence include the chemokine receptor CXCR4, which is known to enable CRC metastasis, as well as the cancer-initiating cell marker and peptidase CD26, which terminates activity of its chemokine CXCL12. METHODS: We evaluated the expression and function of CXCR4 and CD26 in colon cancer cell lines and xenografts following treatment with common chemotherapies using radioligand binding, flow cytometry, immunofluorescence, and enzymatic assays. RESULTS: 5-Fluorouracil, oxaliplatin and SN-38 (the active metabolite of irinotecan), as well as cisplatin, methotrexate and vinblastine, each caused decreases in cell-surface CXCR4 and concomitant increases in CD26 on HT-29, T84, HRT-18, SW480 and SW620 CRC cell lines. Flow cytometry indicated that the decline in CXCR4 was associated with a significant loss of CXCR4+/CD26- cells. Elevations in CD26 were paralleled by increases in both the intrinsic dipeptidyl peptidase activity of CD26 as well as its capacity to bind extracellular adenosine deaminase. Orthotopic HT-29 xenografts treated with standard CRC chemotherapeutics 5-fluorouracil, irinotecan, or oxaliplatin showed dramatic increases in CD26 compared to untreated tumors. Consistent with the loss of CXCR4 and gain in CD26, migratory responses to exogenous CXCL12 were eliminated in cells pretreated with cytotoxic agents, although cells retained basal motility. Analysis of cancer-initiating cell CD44 and CD133 subsets revealed drug-dependent responses of CD26/CD44/CD133 populations, suggesting that the benefits of combining standard chemotherapies 5-fluoruracil and oxaliplatin may be derived from their complementary elimination of cell populations. CONCLUSION: Our results indicate that conventional anticancer agents may act to inhibit chemokine-mediated migration through eradication of CXCR4+ cells and attenuation of chemokine gradients through elevation of CD26 activity.

A non-inflammatory form of immune competence prevails in acute pre-pubescent malnutrition: new evidence based on critical mRNA transcripts in the mouse.

The declining inflammatory immune competence of acute (i.e. wasting) pre-pubescent protein-energy malnutrition has been regarded as reflecting an unregulated immunological disintegration. Recent evidence, however, suggests that malnutrition stimulates a regulated immunological reconfiguration to achieve a non-inflammatory form of competence, perhaps offering protection against autoimmune reactions - the 'Tolerance Model'. Our objective was to determine the influence of acute pre-pubescent malnutrition on the expression of genes critical to tolerogenic regulation. Male and female C57BL/6J mice, initially 19 d old, consumed a complete purified diet either ad libitum (age-matched controls) or in restricted daily quantities (mimicking marasmus), or consumed an isoenergetic low-protein diet ad libitum (mimicking incipient kwashiorkor) for 14 d (six animals per dietary group). Gene expression in the spleen, typically an inflammatory organ, and in the small intestine, a site designed for non-inflammatory defence, was assessed by real-time quantitative RT-PCR, and normalised to ß-actin. In the spleen of the malnourished groups, both IL-10 and transforming growth factor-ß1 mRNA expression increased compared with controls (P < 0.05), whereas mRNA expression of IL-12p40 decreased (P < 0.05). Conversely, malnutrition exerted no influence on the expression of mRNA for these cytokines in the small intestine (P>0.05). Moreover, forkhead box P3 mRNA expression, indicative of cell-based tolerogenic potential, was sustained in both the spleen and intestine of the malnourished groups (P>0.05). Thus, despite limited supplies of energy and substrates, the spleen shifted towards a non-inflammatory character and the intestine was sustained in this mode in advanced pre-pubescent weight loss. These findings provide the first support for the Tolerance Model at the level of mRNA transcript expression.

Nuclear translocation of the 1,25D3-MARRS (membrane associated rapid response to steroids) receptor protein and NFkappaB in differentiating NB4 leukemia cells.

1,25 Dihydroxyvitamin D(3) (1,25D(3)) primes NB4 promyelocytic leukemia cells to differentiate along the monocyte/macrophage lineage through a non-genomic mechanism. Here we show that NB4 cells express high levels of the recently identified membrane receptor for 1,25D(3), which is a distinct gene product from the classical nuclear vitamin D receptor. This 57 kDa protein, named 1,25D(3)-MARRS (Membrane Activated Rapid Response to Steroids)/ERp57/PIA3 appears to associate in a complex with the transcription factor, nuclear factor kappa B (NFkappaB). In unstimulated cells, 1,25D(3)-MARRS can be co-immunoprecipitated with antibodies directed at NFkappaB, and NFkappaB is co-precipitated when antibodies against 1,25D(3)-MARRS or ERp57 are used. Confocal microscopy and subcellular fractionation studies demonstrate that both 1,25D(3)-MARRS and NFkappaB begin translocating to the nucleus within minutes of co-stimulation with 1,25D(3) and phorbol ester. The predominant nuclear localization of both proteins precedes the expression of the monocyte/macrophage phenotype and suggests that this event may be critical to the differentiation pathway. This suggests a role for 1,25D(3)-MARRS in the nucleus as a regulator of gene expression. Here it may also regulate the activity of NFkappaB and other factors with which it may be interacting.

Involvement of 1,25D3-MARRS (membrane associated, rapid response steroid-binding), a novel vitamin D receptor, in growth inhibition of breast cancer cells.

In addition to classical roles in calcium homeostasis and bone development, 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] inhibits the growth of several cancer types, including breast cancer. Although cellular effects of 1,25(OH)2D3 traditionally have been attributed to activation of a nuclear vitamin D receptor (VDR), a novel receptor for 1,25(OH)2D3 called 1,25D3-MARRS (membrane-associated, rapid response steroid-binding) protein was identified recently. The purpose of this study was to determine if the level of 1,25D3-MARRS expression modulates 1,25(OH)2D3 activity in breast cancer cells. Relative levels of 1,25D3-MARRS protein in MCF-7, MDA MB 231, and MCF-10A cells were estimated by real-time RT-PCR and Western blotting. To determine if 1,25D3-MARRS receptor was involved in the growth inhibitory effects of 1,25(OH)2D3 in MCF-7 cells, a ribozyme construct designed to knock down 1,25D(3)-MARRS mRNA was stably transfected into MCF-7 cells. MCF-7 clones in which 1,25D3-MARRS receptor expression was reduced showed increased sensitivity to 1,25(OH)2D3 ( IC(50) 56+/-24 nM) compared to controls (319+/-181 nM; P<0.05). Reduction in 1,25D3-MARRS receptor lengthened the doubling time in transfectants treated with 1,25(OH)2D3. Knockdown of 1,25D3-MARRS receptor also increased the sensitivity of MCF-7 cells to the vitamin D analogs KH1060 and MC903, but not to unrelated agents (all-trans retinoic acid, paclitaxel, serum/glucose starvation, or the isoflavone, pomiferin). These results suggest that 1,25D3-MARRS receptor expression interferes with the growth inhibitory activity of 1,25(OH)2D3 in breast cancer cells, possibly through the nuclear VDR. Further research should examine the potential for pharmacological or natural agents that modify 1,25D3-MARRS expression or activity as anticancer agents.

CXCR4 in Cancer and Its Regulation by PPARgamma.

Chemokines are peptide mediators involved in normal development, hematopoietic and immune regulation, wound healing, and inflammation. Among the chemokines is CXCL12, which binds principally to its receptor CXCR4 and regulates leukocyte precursor homing to bone marrow and other sites. This role of CXCL12/CXCR4 is "commandeered" by cancer cells to facilitate the spread of CXCR4-bearing tumor cells to tissues with high CXCL12 concentrations. High CXCR4 expression by cancer cells predisposes to aggressive spread and metastasis and ultimately to poor patient outcomes. As well as being useful as a marker for disease progression, CXCR4 is a potential target for anticancer therapies. It is possible to interfere directly with the CXCL12:CXCR4 axis using peptide or small-molecular-weight antagonists. A further opportunity is offered by promoting strategies that downregulate CXCR4 pathways: CXCR4 expression in the tumor microenvironment is modulated by factors such as hypoxia, nucleosides, and eicosanoids. Another promising approach is through targeting PPAR to suppress CXCR4 expression. Endogenous PPARgamma such as 15-deoxy-Delta(12,14)-PGJ(2) and synthetic agonists such as the thiazolidinediones both cause downregulation of CXCR4 mRNA and receptor. Adjuvant therapy using PPARgamma agonists may, by stimulating PPARgamma-dependent downregulation of CXCR4 on cancer cells, slow the rate of metastasis and impact beneficially on disease progression.

15-Deoxy-delta(12,14)-prostaglandin J(2) down-regulates CXCR4 on carcinoma cells through PPARgamma- and NFkappaB-mediated pathways.

The chemokine receptor CXCR4 plays a key role in the metastasis of colorectal cancer and its growth at metastatic sites. Here, we have investigated the mechanisms by which CXCR4 on cancer cells might be regulated by eicosanoids present within the colorectal tumor microenvironment. We show that prostaglandins PGE(2), PGA(2), PGD(2), PGJ(2) and 15dPGJ(2) each down-regulates CXCR4 receptor expression on human colorectal carcinoma cells to differing degrees. The most potent of these were PGD(2) and its metabolites PGJ(2) and 15dPGJ(2). Down-regulation was most rapid with the end-product 15dPGJ(2) and was accompanied by a marked reduction in CXCR4 mRNA. 15dPGJ(2) is known to be a ligand for the nuclear receptor PPARgamma. Down-regulation of CXCR4 was also observed with the PPARgamma agonist rosiglitazone, while 15dPGJ(2)-induced CXCR4 down-regulation was substantially diminished by the PPARgamma antagonists GW9662 and T0070907. These data support the involvement of PPARgamma. However, the 15dPGJ(2) analogue CAY10410, which can act on PPARgamma but which lacks the intrinsic cyclopentenone structure found in 15dPGJ(2), down-regulated CXCR4 substantially less potently than 15dPGJ(2). The cyclopentenone grouping is known to inhibit the activity of NFkappaB. Consistent with an additional role for NFkappaB, we found that the cyclopentenone prostaglandin PGA(2) and cyclopentenone itself could also down-regulate CXCR4. Immunolocalization studies showed that the cellular context was sufficient to trigger a focal nuclear pattern of NFkappaB p50 and that 15dPGJ(2) interfered with this p50 nuclear localization. These data suggest that 15dPGJ(2) can down-regulate CXCR4 on cancer cells through both PPARgamma and NFkappaB. 15dPGJ(2), present within the tumor microenvironment, may act to down-regulate CXCR4 and impact upon the overall process of tumor expansion.

Thiazolidinedione drugs down-regulate CXCR4 expression on human colorectal cancer cells in a peroxisome proliferator activated receptor gamma-dependent manner.

Peroxisome proliferator activated receptor (PPAR) gamma is a nuclear receptor involved primarily in lipid and glucose metabolism. PPARgamma is also expressed in several cancer types, and has been suggested to play a role in tumor progression. PPARgamma agonists have been shown to reduce the growth of colorectal carcinoma cells in culture and in xenograft models. Furthermore, the PPARgamma agonist thiazolidinedione has been shown to reduce metastasis in a murine model of rectal cancer. Since the chemokine receptor CXCR4 has emerged as an important player in tumorigenesis, particularly in the process of metastasis, we sought to determine if PPARgamma agonists might act in part by reducing CXCR4 expression. We found that rosiglitazone, a thiazolidinedione PPARgamma agonist used primarily in the treatment of type 2 diabetes, significantly reduced cell-surface expression of CXCR4 protein on HT-29 human colorectal carcinoma cells. This effect occurred at concentrations as low as 1 nM, and was first evident after 8 h of drug exposure. CXCR4 mRNA was also down-regulated after treatment with rosiglitazone, indicating that the effect occurs at the level of transcription. Four other thiazolidinedione compounds (ciglitazone, pioglitazone, troglitazone, and MCC555) also significantly reduced CXCR4 expression. To confirm the involvement of PPARgamma in thiazolidinedione-induced CXCR4 down-regulation, we used PPARgamma antagonists GW9662 and T0070907, both of which completely blocked the effect of rosiglitazone on CXCR4 expression. Furthermore, HT-29 cells in which PPARgamma expression was reduced using shRNA were less responsive to rosiglitazone. In conclusion, we have shown that thiazolidinedione compounds reduce CXCR4 mRNA and cell-surface protein expression in a PPARgamma-dependent manner.

Adenosine upregulates CXCR4 and enhances the proliferative and migratory responses of human carcinoma cells to CXCL12/SDF-1alpha.

The level of expression of the chemokine receptor CXCR4 has been shown to play a crucial role in determining the ability of cancer cells to metastasize from the primary tumor and become established in tissue sites that are rich in the CXCR4 ligand CXCL12/SDF-1alpha. High CXCR4 expression on cancer cells is associated with an increased risk of recurrence and poorer overall survival. We propose that local tissue mediators within the primary tumor or at secondary sites may modulate the level of CXCR4 expression and, therefore, potentially affect the ability of the cancer cells to metastasize. The purine nucleoside adenine-9-beta-D-ribofuranoside (adenosine) is generated at high concentrations within the extracellular fluid of solid tumors because of their hypoxia. We show here that adenosine acts through A(2A) and A(2B) adenosine receptors on human colorectal carcinoma cells to upregulate CXCR4 mRNA expression up to 10-fold and selectively increases cell-surface CXCR4 protein up to 3-fold. This increase in cell-surface CXCR4 enables the carcinoma cells to migrate toward CXCL12, and enhances their proliferation in response to CXCL12. Adenosine may therefore be one of the factors within the tumor microenvironment that facilitates tumor dissemination, by upregulating CXCR4 on certain cancer cells and enhancing cellular responses to CXCL12.