Cancer cell CCR2 orchestrates suppression of the adaptive immune response.

C-C chemokine receptor type 2 (CCR2) is expressed on monocytes and facilitates their recruitment to tumors. Though breast cancer cells also express CCR2, its functions in these cells are unclear. We found that Ccr2 deletion in cancer cells led to reduced tumor growth and approximately twofold longer survival in an orthotopic, isograft breast cancer mouse model. Deletion of Ccr2 in cancer cells resulted in multiple alterations associated with better immune control: increased infiltration and activation of cytotoxic T lymphocytes (CTLs) and CD103+ cross-presenting dendritic cells (DCs), as well as up-regulation of MHC class I and down-regulation of checkpoint regulator PD-L1 on the cancer cells. Pharmacological or genetic targeting of CCR2 increased cancer cell sensitivity to CTLs and enabled the cancer cells to induce DC maturation toward the CD103+ subtype. Consistently, Ccr2-/- cancer cells did not induce immune suppression in Batf3-/- mice lacking CD103+ DCs. Our results establish that CCR2 signaling in cancer cells can orchestrate suppression of the immune response

Cancer cell CCR2 orchestrates suppression of the adaptive immune response

ABSTRACT C-C chemokine receptor type 2 (CCR2) is expressed on monocytes and facilitates their recruitment to tumors. Although breast cancer cells also express CCR2, its functions in these cells are unclear. We found that Ccr2 deletion in cancer cells led to reduced tumor growth and ∼2-fold longer survival in an orthotopic isograft breast cancer mouse model. Deletion of Ccr2 in cancer cells resulted in multiple alterations associated with better immune control: increased infiltration and activation of cytotoxic T lymphocytes (CTLs) and CD103+ cross-presenting dendritic cells (DCs), as well as upregulation of MHC class I and downregulation of checkpoint regulator PD-L1 on the cancer cells. Pharmacological inhibition of CCR2 increased cancer cell sensitivity to CTLs and enabled cancer cells to induce DC maturation toward the CD103+ subtype. Consistently, Ccr2 -/- cancer cells did not induce immune suppression in Batf3 -/- mice lacking the CD103+ DC subtype. Our results establish that CCR2 signaling in cancer cells can orchestrate suppression of the immune response. Summary C-C chemokine receptor type 2 (CCR2) expressed on monocytes facilitates their recruitment to tumors. Here, CCR2 signaling in cancer cells is shown to suppress immune control of tumors, in part by reducing CD103+ dendritic cell recruitment

Multi-color Flow Cytometry for Comprehensive Analysis of the Tumor Immune Infiltrate in a Murine Model of Breast Cancer.

Flow cytometry is a popular laser-based technology that allows the phenotypic and functional characterization of individual cells in a high-throughput manner. Here, we describe a detailed procedure for preparing a single-cell suspension from mammary tumors of the mouse mammary tumor virus-polyoma middle T (MMTV-PyMT) and analyzing these cells by multi-color flow cytometry. This protocol can be used to study the following tumor-infiltrating immune cell populations, defined by the expression of cell surface molecules: total leukocytes, tumor-associated macrophages (TAMs), conventional dendritic cells (DCs), CD103-expressing DCs, tumor-associated neutrophils, inflammatory monocytes, natural killer (NK) cells, CD4+ T cells, CD8+ T cells, γδT cells, and regulatory T cells

Passive leakage across monolayers using calcein as a fluorescent marker

<p>This dataset tabulates data on passive leakage across the monolayers using a fluorescent marker, calcein, a derivative of fluorescein. The purpose was to gauge the degree of confluence and whether tight junctions had formed between the epithelial cells.</p> <p>The sheet 'Leak rates' tabulates a summary of the data of the 17 calcein experiments in the raw data sheet. The sheet 'Raw data' contains the data and calculations of the 17 experiments.</p

Tests for nonspecific protein effect by exposure to bovine serum albumin (BSA)

<p>This dataset contains the data and calculations for 7 control experiments which tested for a nonspecific protein effect by measuring the effects on sodium transepithelial movement of exposing cultures to bovine serum albumin (BSA), rather than urinary precipitates.<br>The 'Summary' sheet summarizes the results of the experiments. The other 7 sheets (named by date of experiment) contain the raw data and calculations for the 7 experiments. The effect on sodium transport was calculated in the same way as in the experiments utilizing urinary precipitates.</p> <p> </p

Transepithelial sodium transport in neurosurgical patients with renal salt wasting

<p>This dataset contains the results of experiments measuring the transport of sodium across the monolayers. 22Na was used to make these measurements. The experiments tested the effects of resuspended urinary precipitates from renal salt wasting (RSW) patients and non-RSW Control patients. The amount of protein in the precipitates was used to quantitate the precipitates.</p> <p>The 'Summary' sheet tabulates the results of the individual experiments described below. The heading numbers such as 5ug or 10ug refer to the concentrations of resuspended precipitates (in micrograms protein/ml) to which the cultures were exposed. In all experiments, cultures were exposed to precipitates on both the mucosal and serosal sides. Numbers tabulated are the effects of various concentrations of precipitate on transepithelial Na movement, expressed as percentages relative to that seen side-by-side in vehicle-only wells.</p> <p>The control and RSW sheets contain the raw data and calculations, for 47 individual experiments in which the effects of precipitates from the urine of RSW-afflicted patients or non-RSW Control patients on sodium transport across cultured LLC monolayers were measured. Effects of the precipitates on sodium movements were calculated as described in the Methods section of the paper, in brief, the effect of treatment (change in the slope of the linear regression line of sodium movement after treatment versus before) in experimental culture wells versus the effect of vehicle treatment (change in the regression line after versus before treatment) seen in side-by-side Vehicle wells. The results for individual patients are grouped in sub-folders with the patients identified by index numbers.</p> <p>Other numbers quoted are percent change in transepithelial sodium transport (caused by addition of urinary precipitate, indexed by its protein content) relative to vehicle addition, as described in Methods in body of the paper. (Briefly, relative to Na movement in side-by-side culture wells in which only vehicle was added to the transport medium).</p

Double-blind, randomized placebo controlled trial of fulvestrant compared with exemestane after prior nonsteroidal aromatase inhibitor therapy in postmenopausal women with hormone receptor-positive, advanced breast cancer: results from EFECT.

PURPOSE: The third-generation nonsteroidal aromatase inhibitors (AIs) are increasingly used as adjuvant and first-line advanced therapy for postmenopausal, hormone receptor-positive (HR+) breast cancer. Because many patients subsequently experience progression or relapse, it is important to identify agents with efficacy after AI failure. MATERIALS AND METHODS: Evaluation of Faslodex versus Exemestane Clinical Trial (EFECT) is a randomized, double-blind, placebo controlled, multicenter phase III trial of fulvestrant versus exemestane in postmenopausal women with HR+ advanced breast cancer (ABC) progressing or recurring after nonsteroidal AI. The primary end point was time to progression (TTP). A fulvestrant loading-dose (LD) regimen was used: 500 mg intramuscularly on day 0, 250 mg on days 14, 28, and 250 mg every 28 days thereafter. Exemestane 25 mg orally was administered once daily. RESULTS: A total of 693 women were randomly assigned to fulvestrant (n = 351) or exemestane (n = 342). Approximately 60% of patients had received at least two prior endocrine therapies. Median TTP was 3.7 months in both groups (hazard ratio = 0.963; 95% CI, 0.819 to 1.133; P = .6531). The overall response rate (7.4% v 6.7%; P = .736) and clinical benefit rate (32.2% v 31.5%; P = .853) were similar between fulvestrant and exemestane respectively. Median duration of clinical benefit was 9.3 and 8.3 months, respectively. Both treatments were well tolerated, with no significant differences in the incidence of adverse events or quality of life. Pharmacokinetic data confirm that steady-state was reached within 1 month with the LD schedule of fulvestrant. CONCLUSION: Fulvestrant LD and exemestane are equally active and well-tolerated in a meaningful proportion of postmenopausal women with ABC who have experienced progression or recurrence during treatment with a nonsteroidal AI.Clinical Trial, Phase IIIJournal ArticleMulticenter StudyRandomized Controlled Trialinfo:eu-repo/semantics/publishe

Cancer cells induce metastasis-supporting neutrophil extracellular DNA traps

Neutrophils, the most abundant type of leukocytes in blood, can form neutrophil extracellular traps (NETs). These are pathogen-trapping structures generated by expulsion of the neutrophil's DNA with associated proteolytic enzymes. NETs produced by infection can promote cancer metastasis. We show that metastatic breast cancer cells can induce neutrophils to form metastasis-supporting NETs in the absence of infection. Using intravital imaging, we observed NET-like structures around metastatic 4T1 cancer cells that had reached the lungs of mice. We also found NETs in clinical samples of triple-negative human breast cancer. The formation of NETs stimulated the invasion and migration of breast cancer cells in vitro. Inhibiting NET formation or digesting NETs with deoxyribonuclease I (DNase I) blocked these processes. Treatment with NET-digesting, DNase I-coated nanoparticles markedly reduced lung metastases in mice. Our data suggest that induction of NETs by cancer cells is a previously unidentified metastasis-promoting tumor-host interaction and a potential therapeutic target