Interaction between CXCR4 and CCL20 Pathways Regulates Tumor Growth

The chemokine receptor CXCR4 and its ligand CXCL12 is overexpressed in the majority of tumors and is critically involved in the development and metastasis of these tumors. CXCR4 is expressed in malignant tumor cells whereas its ligand SDF-1 (CXCL12) is expressed mainly by cancer associated fibroblasts (CAF). Similarly to CXCR4, the chemokine CCL20 is overexpressed in variety of tumors; however its role and regulation in tumors is not fully clear. Here, we show that the chemokine receptor CXCR4 stimulates the production of the chemokine CCL20 and that CCL20 stimulates the proliferation and adhesion to collagen of various tumor cells. Furthermore, overexpression of CCL20 in tumor cells promotes growth and adhesion in vitro and increased tumor growth and invasiveness in vivo. Moreover, neutralizing antibodies to CCL20 inhibit the in vivo growth of tumors that either overexpress CXCR4 or CCL20 or naturally express CCL20. These results reveal a role for CCL20 in CXCR4-dependent and -independent tumor growth and suggest a therapeutic potential for CCL20 and CCR6 antagonists in the treatment of CXCR4- and CCL20-dependent malignancies

Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

CCL20, CCR6 and CXCR4 expression in prostate cancer cell lines, in primary prostate tumor tissue and in normal prostate tissue.

<p>(A) Expression of CCL20 and CCR6 in human prostate cancer tissue and in human normal prostate tissue. Immunohistostaining of prostate cancer and normal specimens using the polyclonal antibody for CCL20 and the monoclonal antibody 140706 for CCR6. Original magnification of ×400 is shown. CCL20 and CCR6 expression was observed in endothelial and fibromuscular cells of prostate samples (signed with black arrows). A commercially available array including 52 samples (CA2) from patients with prostate cancer was stained for CCL20 (A′) and CCR6 (A″). Expression was scored on two levels: low or negative expression, and high expression. Statistical analysis of the immunohistochemical staining was performed using two-tailed Mann-Whitney test. (B) Comnmon expression pattern of CCL20 and CXCR4 in human prostate cancer. Immunohistostaining of prostate cancer specimens using the polyclonal antibody for CCL20 and the monoclonal antibody 12G5 for CXCR4. Original magnification of ×400 is shown. CCL20 and CXCR4 expression was observed in endothelial and fibromuscular cells of prostate samples. (B′) A commercially available array including 48 samples (CA3) from patients with prostate cancer was stained for CCL20 and CXCR4. Expression was scored on three levels: low, intermediate, and high expression. Samples that demonstrated the same scoring in CCL20 and CXCR4 expression levels were classified as having similar expression pattern. Samples where scores in CCL20 and CXCR4 expression differed in one level or more were classified as different expression patterns.</p

Regulation of CCL20 expression and function.

<p>(A–B) CXCR4 expression levels of PC3-CXCR4.5 cells 48 hours following the transfection with control non-specific siRNA and specific anti-CXCR4 siRNA. The cells were stained for the control and CXCR4 antibodies and evaluated by FACS. Semi-quantitative RT-PCR analysis of CXCR4 and CCL20 mRNA of the siRNA-transfected PC3-CXCR4.5 cells 48 hours following the transfection. β-actin confirmed comparable loading of RT-PCR products in each lane. Control and CXCR4 siRNA-transfected PC3-CXCR4.5 cells at 48 hours post-transfection were incubated with CXCL12 500 ng/ml for an additional 48 hours. CCL20 secretion was assessed by ELISA. The results represent the average of triplicates±SD (** <i>P</i><0.05). (C) CCR6 expression in prostate cancer cell lines PC3, LaNCAP, 22Rv1 and DU145 was evaluated by FACS and PCR whereas CCL20 expresion was evaluated by PCR and ELISA assays. Purple line represents mouse IgG control antibody, green line represents staining with CCR6 monoclonal antibody. (D) PC3 and PC3-CXCR4 cells were incubated with various concentration of CCL20 for 6 days. Following 3 days of incubation, the medium with or without CCL20 was renewed. On day 6, the cells were harvested and viable cells were counted using PI staining and FACS analysis. In addition, in order to determine proliferation of PC3 cells, the cells were labeled with BrdU (10 µM) during the last 16 hours of incubation and processed for BrdU detection using specific anti-BrdU FITC-conjugated antibody and FACS analysis. Data is presented as mean±SD from triplicates (** <i>P</i><0.05). Data is representative of two separate experiments. (E–F) PC3 and PC3-CXCR4.5 cells either untreated or treated with various concentrations of CCL20 were placed on collagen I- or fibronectin-coated plates (10 µg/ml) for 30 minutes. Non-adherent cells were washed twice with cold PBS. Adherent cells were collected in 300 µl FACS buffer with 5 mM EDTA and counted by FACS. Data is presented as mean±SD from triplicates (** <i>P</i><0.05). In addition, PC3 cells (E) that demonstrated increased adhesion to collagen I and fibronectin in response to stimulation with CCL20, were co-incubated with CCL20 and PTX (100 ng/ml) and were allowed to adhere to collagen I- and fibronectin-coated plates.</p

CCL20 regulates CXCR4 dependent and independent growth of tumor cells.

<p>(A) Effect of CCL20 stable expression on prostate tumor growth. PC3-CCL20.30, PC3-CCL20.10 and PC3-mock transfected cells (5×10⁶/mouse) were injected subcutaneously into SCID/beige mice. 48 days or 75 days following the injection, animals were sacrificed, and xenograft tumors generated by PC3-mock and PC3-CCL20.30 injected cells were purified . Tumor size (cm²) was measured twice a week using caliper. Results are representative of three independent experiments with five mice in each group. Data is presented as mean±SE from five mice. (B) H&E staining of paraffin-embedded tumor tissue sections derived from PC3-mock and PC3-CCL20.30 tumors on day 48. Black arrows signify non-invasive borders of PC3-mock tumor (B,a), small blood vessel in PC3-mock tumor (B,b), aberrant blood vessels in PC3-CCL20.30 tumor (B, c,d), original magnification of ×200 is shown. (C) Vessel functionality (Δ<i>S</i>o₂) was measured by fMRI. Functionality of the vasculature was tested during inhalation of air-CO₂ and carbogen (95% oxygen+5% CO₂) in mice implanted with PC3-mock cells or with PC3-CCL20.30 cells. Δ<i>S</i>o₂ values from PC3-mock cells and PC3-CCL20.30 are shown. The mean±SD values of Δ<i>S</i>o₂ were calculated from whole tumor, and normalized to contra-lateral muscle, pooling data from 9 mice from the PC3-CCL20.30 group and 5 mice from the PC3-mock group (four slices/mouse; p<0.001). (D) Adhesion of PC3-CCL20.30 cells to collagen I. PC3-CCL20.30 cells either umstimulated or stimulated with 50 ng/ml of CCL20 with or without co-incubation with neutralizing anti-CCL20 antibodies (10 µg/ml) were placed on collagen I-coated plates (10 µg/ml) for 30 minutes. Non-adherent cells were washed twice with cold PBS. Adherent cells were collected in 300 µl FACS buffer with 5 mM EDTA and counted by FACS. Data is presented as mean±SD from triplicates (** <i>P</i><0.016). (E) PC3-CCL20.30 cells (5×10⁶/mouse) were injected subcutaneously into SCID/beige mice. Twenty-four hours after the cell injection, mice started to get subcutaneous injections of anti-human CCL20 antibodies or isotype control antibodies, 20 µg of antibody per injection, three times a week, during four weeks. Tumor size (cm²) was measured once a week using caliper. Results are representative of two independent experiments with ten mice in each group. Data is presented as mean±SE from ten mice. (F) On day 64 following the cell injection, the experiment was terminated, animals were sacrificed; subcutaneous tumors were removed, measured and weighted. Data is presented as mean±SE from ten mice in each group (** <i>P</i><0.0002). (G) PC3-CXCR4.5 cells (5×10⁶/mouse) were injected subcutaneously into SCID/beige mice. Twenty-four hours after the cell injection, mice started to get subcutaneous injections of anti-human CCL20 antibodies or isotype control antibodies, 20 µg of antibody per injection, three times a week, during four weeks. Tumor size (cm²) was measured once a week using caliper. On day 55 following cell injection, the experiment was terminated; animals were sacrificed, subcutaneous tumors were removed, measured and weighted. Data is presented as mean±SE from ten mice in each group (** <i>P</i><0.0027). (H) HT-29 cells (2×10⁶/mouse) were injected subcutaneously into nude mice. Twenty-four hours after cell injection, mice started to get subcutaneous injections of anti-human CCL20 antibodies or isotype control antibodies, 20 µg of antibody per injection, five times a week, during two weeks. On day 17 following cell injection, the experiment was terminated, animals were sacrificed; subcutaneous tumors were removed, measured and weighted. Data is presented as mean±SE from ten mice in each group (** <i>P</i><0.0002).</p