CCL2 recruits inflammatory monocytes to facilitate breast-tumour metastasis.

Macrophages, which are abundant in the tumour microenvironment, enhance malignancy. At metastatic sites, a distinct population of metastasis-associated macrophages promotes the extravasation, seeding and persistent growth of tumour cells. Here we define the origin of these macrophages by showing that Gr1-positive inflammatory monocytes are preferentially recruited to pulmonary metastases but not to primary mammary tumours in mice. This process also occurs for human inflammatory monocytes in pulmonary metastases of human breast cancer cells. The recruitment of these inflammatory monocytes, which express CCR2 (the receptor for chemokine CCL2), as well as the subsequent recruitment of metastasis-associated macrophages and their interaction with metastasizing tumour cells, is dependent on CCL2 synthesized by both the tumour and the stroma. Inhibition of CCL2-CCR2 signalling blocks the recruitment of inflammatory monocytes, inhibits metastasis in vivo and prolongs the survival of tumour-bearing mice. Depletion of tumour-cell-derived CCL2 also inhibits metastatic seeding. Inflammatory monocytes promote the extravasation of tumour cells in a process that requires monocyte-derived vascular endothelial growth factor. CCL2 expression and macrophage infiltration are correlated with poor prognosis and metastatic disease in human breast cancer. Our data provide the mechanistic link between these two clinical associations and indicate new therapeutic targets for treating metastatic breast cancer.

Single cells from human primary colorectal tumors exhibit polyfunctional heterogeneity in secretions of ELR+ CXC chemokines.

Cancer is an inflammatory disease of tissue that is largely influenced by the interactions between multiple cell types, secreted factors, and signal transduction pathways. While single-cell sequencing continues to refine our understanding of the clonotypic heterogeneity within tumors, the complex interplay between genetic variations and non-genetic factors ultimately affects therapeutic outcome. Much has been learned through bulk studies of secreted factors in the tumor microenvironment, but the secretory behavior of single cells has been largely uncharacterized. Here we directly profiled the secretions of ELR+ CXC chemokines from thousands of single colorectal tumor and stromal cells, using an array of subnanoliter wells and a technique called microengraving to characterize both the rates of secretion of several factors at once and the numbers of cells secreting each chemokine. The ELR+ CXC chemokines are highly redundant, pro-angiogenic cytokines that signal via the CXCR1 and CXCR2 receptors, influencing tumor growth and progression. We find that human primary colorectal tumor and stromal cells exhibit polyfunctional heterogeneity in the combinations and magnitudes of secretions for these chemokines. In cell lines, we observe similar variance: phenotypes observed in bulk can be largely absent among the majority of single cells, and discordances exist between secretory states measured and gene expression for these chemokines among single cells. Together, these measures suggest secretory states among tumor cells are complex and can evolve dynamically. Most importantly, this study reveals new insight into the intratumoral phenotypic heterogeneity of human primary tumors.

The orl rat with inherited cryptorchidism has increased susceptibility to the testicular effects of in utero dibutyl phthalate exposure.

Phenotype results from interactions between genetics and environment, but for most environmental chemical exposures, such interactions are theoretical. The phenotypic response of the testis to in utero dibutyl phthalate (DBP) exposure was compared between two strains of Long-Evans (LE) rats, the orl substrain with inherited cryptorchidism and an outbred (wt) strain. orl and wt LE rats were exposed daily between gestational day (GD) 12 and GD21 to DBP dose levels ranging from 50 to 200 mg/kg by oral gavage and sensitive phthalate testicular end points examined at either GD19, GD21, or postnatal day (PND) 21. At 50 mg/kg DBP, GD19 expression of Cyp17a1, Insl3, and Scarb1 was significantly reduced in orl but not wt testis. At GD21, statistically significant differential strain effects (orl more sensitive than wt) were observed for testicular expression of Scarb1 at 50 and 200 mg/kg DBP and Star at 200 mg/kg DBP. Similarly, DBP exposure disproportionately increased GD21 seminiferous cord diameters and numbers of multinucleated germ cells in the orl strain. At PND21, body weight-corrected testis weights were lowered significantly by DBP exposure at all dose levels in the orl strain but not in wt rats. While the frequency of undescended testes after 200 mg/kg DBP exposure in the orl strain appeared increased, these data were not statistically significant. These results demonstrated enhanced sensitivity of the orl rat to phthalate exposure as compared to its parent strain, a potentially important model of the effects of gene-environment interaction on development of male reproductive malformations.

Altered expression of muscle- and cytoskeleton-related genes in a rat strain with inherited cryptorchidism.

Development of the fetal gubernaculum is a prerequisite for testicular descent and dependent on insulin-like 3 and androgen, but knowledge of downstream effectors is limited. We analyzed transcript profiles in gubernaculum and testis to address changes occurring during normal and abnormal testicular descent in Long Evans wild-type (wt) and cryptorchid (orl) fetuses. Total RNA from male wt and orl gubernacula (gestational days [GD]18-20), wt female gubernacula (GD18), and testis (GD17 and 19) was hybridized to Affymetrix GeneChips. Statistical analysis of temporal, gender, and strain-specific differences in gene expression was performed with the use of linear models analysis with empirical Bayes statistics and analysis of variance (gubernaculum) and linear analysis (testis). Overrepresented common gene ontology functional categories and pathways were identified in groups of differentially expressed genes with the Database for Annotation, Visualization, and Integrated Discovery. Transcript profiles were dynamic in wt males between GD18-19 and GD20, comparatively static in orl GD18-20 gubernaculum, and similar in wt and orl testis. Functional analysis of differentially expressed genes in wt and orl gubernaculum identified categories related to metabolism, cellular biogenesis, small GTPase-mediated signal transduction, cytoskeleton, muscle development, and insulin signaling. Genes involved in androgen receptor signaling, regulated by androgens, or both were overrepresented in differentially expressed gubernaculum and testis gene groups. Quantitative reverse transcription polymerase chain reaction (RT-PCR) confirmed differential expression of genes related to muscle development, including Myog, Tnnt2, Fst, Igf1, Igfbp5, Id2, and Msx1. These data suggest that the orl mutation results in a primary gubernacular defect that affects muscle development and cytoskeletal function and might alter androgen-regulated pathways.

Failure of shortening and inversion of the perinatal gubernaculum in the cryptorchid long-evans orl rat.

PURPOSE: Failure of testicular descent occurs in about 65% of spontaneously cryptorchid Long-Evans orl rats. Development of the fetal gubernaculum is dependent on expression of leucine rich G protein coupled receptor (also known as G protein coupled receptor affecting testicular descent) and its ligand, insulin-like 3. We studied testicular descent and mRNA expression of insulin-like 3 and leucine rich G protein coupled receptor in Long-Evans orl and Long-Evans wild-type rats during perinatal development. MATERIALS AND METHODS: Long-Evans orl and Long-Evans wild-type males obtained at gestational days 18 to 21 and day of birth were preserved in RNAlater for at least 24 hours. The size and position of the testes, kidneys and gubernacula were determined by microdissection and image analysis. Leucine rich G protein coupled receptor mRNA expression was analyzed in fetal gubernacula (gestational days 18 to 21) using real-time reverse transcriptase polymerase chain reaction with SYBR Green detection. Insulin-like 3 mRNA expression in fetal testis (gestational days 18 and 20) was determined by semiquantitative reverse transcriptase polymerase chain reaction. RESULTS: Testicular position was similar in Long-Evans orl and Long-Evans wild-type fetal rats. However, gubernacula were narrow (p < 0.001) and elongated (p < or = 0.01) in Long-Evans orl compared to Long-Evans wild-type fetuses at all time points. Inversion of both gubernacula occurred by the day of birth in 45% of Long-Evans orl and 100% of Long-Evans wild-type males (p < 0.001). Leucine rich G protein coupled receptor mRNA expression decreased with age and, similar to insulin-like 3 mRNA, was not consistently different between strains. CONCLUSIONS: These data suggest that the impaired shortening of the Long-Evans orl gubernaculum may interfere with the timing and quality of its inversion at birth, leading to failure of testicular descent in some newborn males. However, fetal testicular descent and the expression of leucine rich G protein coupled receptor and insulin-like 3 mRNA appear to be normal in this strain.