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Supplemental Table 3 Race and Survival Predictor Regulon Gene Enrichment Ontology Processes

Supplemental Figure 5 Overall breast cancer survival based on median income of county of diagnosis in European American (EA) (A) compared to African American (AA) patients (B). CI, confidence interval.

Supplemental Table 1 Survival and Race Predictor Regulon Gene Descriptions & Regulons

Supplemental Table 2 Race and 3-year Survival Regulon RFS

Supplemental Figure 4 Quantitative correlation of biomarker expression stratified by race.

Supplemental Figure 3 Racial difference in survival in (A) Luminal A compared to (B) TNBC breast cancer. CI, confidence interval.

Supplemental Figure 1 Comparison of subtype distribution by ancestry in the current cohort study

Purpose:Compared with their European American (EA) counterparts, African American (AA) women are more likely to die from breast cancer in the United States. This disparity is greatest in hormone receptor–positive subtypes. Here we uncover biological factors underlying this disparity by comparing functional expression and prognostic significance of master transcriptional regulators of luminal differentiation.Experimental Design:Data and biospecimens from 262 AA and 293 EA patients diagnosed with breast cancer from 2001 to 2010 at a major medical center were analyzed by IHC for functional biomarkers of luminal differentiation, including estrogen receptor (ESR1) and its pioneer factors, FOXA1 and GATA3. Integrated comparison of protein levels with network-level gene expression analysis uncovered predictive correlations with race and survival.Results:Univariate or multivariate HRs for overall survival, estimated from digital IHC scoring of nuclear antigen, show distinct differences in the magnitude and significance of these biomarkers to predict survival based on race: ESR1 [EA HR = 0.47; 95% confidence interval (CI), 0.31–0.72 and AA HR = 0.77; 95% CI, 0.48–1.18]; FOXA1 (EA HR = 0.38; 95% CI, 0.23–0.63 and AA HR = 0.53; 95% CI, 0.31–0.88), and GATA3 (EA HR = 0.36; 95% CI, 0.23–0.56; AA HR = 0.57; CI, 0.56–1.4). In addition, we identify genes in the downstream regulons of these biomarkers highly correlated with race and survival.Conclusions:Even within clinically homogeneous tumor groups, regulatory networks that drive mammary luminal differentiation reveal race-specific differences in their association with clinical outcome. Understanding these biomarkers and their downstream regulons will elucidate the intrinsic mechanisms that drive racial disparities in breast cancer survival.

Supplemental data

To investigate subtype-specific risk of germline alleles associated with triple negative breast cancer (TNBC) in African ancestry populations.Breast cancer (BC) mortality is higher in African American (AA) compared to White American (WA) women; this disparity is partly explained by 2-fold higher TNBC incidence.We used a surgically maintained biospecimen cohort of 2884 BC cases. Subsets of the total (760 AA; 962 WA; 910 West African/Ghanaian; 252 East African/Ethiopian) were analyzed for genotypes of candidate alleles. A subset of 417 healthy controls were also genotyped, to measure associations with overall BC risk and TNBC.TNBC frequency was highest in Ghanaian and AA cases (49% and 44% respectively; P0.0001) and lowest in Ethiopian and WA cases (17% and 24% respectively; P0.0001). TNBC cases had higher West African ancestry than non-TNBC (P0.0001). Frequency of the Duffy-null allele (rs2814778; an African ancestral variant adopted under selective pressure as protection against malaria) was associated with TNBC-specific risk (P0.0001), quantified West African Ancestry (P0.0001) and was more common in AA, Ghanaians, and TNBC cases. Additionally, rs4849887 was significantly associated with overall BC risk, and both rs2363956 and rs13000023 were associated with TNBC-specific risk, although none as strongly as the Duffy-null variant.West African ancestry is strongly correlated with TNBC status, as well as germline variants related to BC risk. The Duffy-null allele was associated with TNBC risk in our cohort.

Background: Tumor-specific immune response is an important aspect of disease prognosis and ultimately impacts treatment decisions for innovative immunotherapies. The atypical chemokine receptor 1 (ACKR1 or DARC) gene plays a pivotal role in immune regulation and harbors several single-nucleotide variants (SNV) that are specific to sub-Saharan African ancestry. Methods: Using computational The Cancer Genome Atlas (TCGA) analysis, case–control clinical cohort Luminex assays, and CIBERSORT deconvolution, we identified distinct immune cell profile–associated DARC/ACKR1 tumor expression and race with increased macrophage subtypes and regulatory T cells in DARC/ACKR1-high tumors. Results: In this study, we report the clinical relevance of DARC/ACKR1 tumor expression in breast cancer, in the context of a tumor immune response that may be associated with sub-Saharan African ancestry. Briefly, we found that for infiltrating carcinomas, African Americans have a higher proportion of DARC/ACKR1-negative tumors compared with white Americans, and DARC/ACKR1 tumor expression is correlated with proinflammatory chemokines, CCL2/MCP-1 (P Conclusions: DARC/AKCR1 regulates immune responses in tumors, and its expression is associated with sub-Saharan African-specific alleles. DARC/ACKR1-positive tumors will have a distinct immune response compared with DARC/AKCR1-negative tumors. Impact: This study has high relevance in cancer management, as we introduce a functional regulator of inflammatory chemokines that can determine an infiltrating tumor immune cell landscape that is distinct among patients of African ancestry.

Tryptophan (Trp) is an essential amino acid primarily derived from the diet for use by the host for protein synthesis. The intestinal tract is lined with cells, both host and microbial, that uptake and metabolize Trp to also generate important signaling molecules. Serotonin (5-HT), kynurenine and its downstream metabolites, and to a lesser extent other neurotransmitters are generated by the host to signal onto host receptors and elicit physiological effects. 5-HT production by neurons in the CNS regulates sleep, mood, and appetite; 5-HT production in the intestinal tract by enterochromaffin cells regulates gastric motility and inflammation in the periphery. Kynurenine can signal onto the aryl hydrocarbon receptor (AHR) to elicit pleiotropic responses from several cell types including epithelial and immune cells, or can be further metabolized into bioactive molecules to influence neurodegenerative disease. There is a remarkable amount of cross-talk with the microbiome with regard to tryptophan metabolites as well. The gut microbiome can regulate the production of host tryptophan metabolites and can use dietary or recycled trp to generate bioactive metabolites themselves. Trp derivatives like indole are able to signal onto xenobiotic receptors, including AHR, to elicit tolerogenic effects. Here, we review studies that demonstrate that tryptophan represents a key intra-kingdom signaling molecule.

Large-scale efforts to identify breast cancer (BC) risk alleles have historically taken place among women of European ancestry. Recently, there are new efforts to verify if these alleles increase risk in African American (AA) women as well. We investigated the effect of previously reported AA breast cancer and triple-negative breast cancer (TNBC) risk alleles in our African-enriched International Center for the Study of Breast Cancer Subtypes (ICSBCS) cohort. Using case–control, case-series and race-nested approaches, we report that the Duffy-null allele (rs2814778) is associated with TNBC risk (OR = 3.814, p = 0.001), specifically among AA individuals, after adjusting for self-indicated race and west African ancestry (OR = 3.368, p = 0.007). We have also validated the protective effect of the minor allele of the ANKLE1 missense variant rs2363956 among AA for TNBC (OR = 0.420, p = 0.005). Our results suggest that an ancestry-specific Duffy-null allele and differential prevalence of a polymorphic gene variant of ANKLE1 may play a role in TNBC breast cancer outcomes. These findings present opportunities for therapeutic potential and future studies to address race-specific differences in TNBC risk and disease outcome.

Intestinal epithelial cells (IEC) are crucial for maintaining proper digestion and overall homeostasis of the gut mucosa. IEC proliferation and differentiation are tightly regulated by well described pathways, however, relatively little is known about how cytokines shape these processes. Given that the anti-inflammatory cytokine interleukin (IL)-10 promotes intestinal barrier function, and insufficient IL-10 signaling increases susceptibility to intestinal diseases like inflammatory bowel disease, we hypothesized that IL-10 signaling modulates processes underlying IEC proliferation and differentiation. This was tested using in vivo and in vitro IEC-specific IL-10 receptor 1 (IL-10R1) depletion under homeostatic conditions. Our findings revealed that loss of IL-10R1 drove lineage commitment toward a dominant goblet cell phenotype while decreasing absorptive cell-related features. Diminished IL-10 signaling also significantly elevated IEC proliferation with relatively minor changes to apoptosis. Characterization of signaling pathways upstream of proliferation demonstrated a significant reduction in the Wnt inhibitor, DKK1, increased nuclear localization of β-catenin, and increased transcripts of the proliferation marker, OLFM4, with IL-10R1 depletion. Phosphorylated STAT3 was nearly completely absent in IL-10R1 knockdown cells and may provide a mechanistic link between our observations and the regulation of these cellular processes. Our results demonstrate a novel role for IL-10 signaling in intestinal mucosal homeostasis by regulating proper balance of proliferation and IEC lineage fate.

BACKGROUND Breast cancer mortality rates are 39% higher in the African-American (AA) women compared to White-American (WA) women despite the advances in overall breast cancer screening and treatments. Several studies have undertaken to identify the factors leading to this disparity in United States with possible effects of lower socioeconomic status and underlying aggressive biology. METHODS A retrospective analysis was done using a prospectively maintained database of a metropolitan health system. Patients were selected based on diagnosis of early-stage breast cancer between 10/1998 and 02/2017, and included women over age of 18 with clinically node-negative disease. Patients were then stratified by phenotype confirmed by pathology and patient-identified race. RESULTS A total of 2,298 women were identified in the cohort with 39% AA and 61% WA women. The overall mean age at the time of diagnosis for AA women was slightly younger at 60 years compared to 62 years for WA women (p = 0.003). Follow-up time was longer for the WA women at 95 months vs. 86 months in AA women. The overall 5-year survival was analyzed for the entire cohort, with the lowest survival occurring in patients with triple-negative breast cancer (TNBC). Phenotype distribution revealed a higher incidence of TNBC in AA women compared to WA women (AA 16% vs. WA 10%; p

Purpose This study characterizes the US clinical genetics workforce to inform workforce planning and public policy development. Methods A 32-question survey was electronically distributed to American Board of Medical Genetics and Genomics board-certified/eligible diplomates in 2019. We conducted a descriptive analysis of responses from practicing clinical geneticists. Results Of the 491 clinical geneticists responding to the survey, a majority were female (59%) and White (79%), worked in academic medical centers (73%), and many engaged in telemedicine (33%). Clinical geneticists reported an average of 13 new and 10 follow-up patient visits per week. The average work week was 50 hours and the majority (58%) worked over half-time in clinical duties. Providers indicated that 39% of new emergency patients wait 3 days or more, and 39% of nonemergency patients wait over 3 months to be seen. Respondents were geographically concentrated in metropolitan areas and many reported unfilled clinical geneticist job vacancies at their institution of more than 3 years. Conclusion With the rapid expansion of genomic medicine in the past decade, there is still a gap between genetics services needed and workforce capacity. A concerted effort is required to increase the number of clinical geneticists and enhance interdisciplinary teamwork to meet increasing patient needs.

Purpose: Compared with their European American (EA) counterparts, African American (AA) women are more likely to die from breast cancer in the United States. This disparity is greatest in hormone receptor–positive subtypes. Here we uncover biological factors underlying this disparity by comparing functional expression and prognostic significance of master transcriptional regulators of luminal differentiation. Experimental Design: Data and biospecimens from 262 AA and 293 EA patients diagnosed with breast cancer from 2001 to 2010 at a major medical center were analyzed by IHC for functional biomarkers of luminal differentiation, including estrogen receptor (ESR1) and its pioneer factors, FOXA1 and GATA3. Integrated comparison of protein levels with network-level gene expression analysis uncovered predictive correlations with race and survival. Results: Univariate or multivariate HRs for overall survival, estimated from digital IHC scoring of nuclear antigen, show distinct differences in the magnitude and significance of these biomarkers to predict survival based on race: ESR1 [EA HR = 0.47; 95% confidence interval (CI), 0.31–0.72 and AA HR = 0.77; 95% CI, 0.48–1.18]; FOXA1 (EA HR = 0.38; 95% CI, 0.23–0.63 and AA HR = 0.53; 95% CI, 0.31–0.88), and GATA3 (EA HR = 0.36; 95% CI, 0.23–0.56; AA HR = 0.57; CI, 0.56–1.4). In addition, we identify genes in the downstream regulons of these biomarkers highly correlated with race and survival. Conclusions: Even within clinically homogeneous tumor groups, regulatory networks that drive mammary luminal differentiation reveal race-specific differences in their association with clinical outcome. Understanding these biomarkers and their downstream regulons will elucidate the intrinsic mechanisms that drive racial disparities in breast cancer survival.

More than 40% of patients with luminal breast cancer treated with endocrine therapy agent tamoxifen demonstrate resistance. Emerging evidence suggests tumor initiating cells (TICs) and aberrant activation of Src and Akt signaling drive tamoxifen resistance and relapse. We previously demonstrated that aryl hydrocarbon receptor ligand aminoflavone (AF) inhibits the expression of TIC gene α6-integrin and disrupts mammospheres derived from tamoxifen-sensitive breast cancer cells. In the current study, we hypothesize that tamoxifen-resistant (TamR) cells exhibit higher levels of α6-integrin than tamoxifen-sensitive cells and that AF inhibits the growth of TamR cells by suppressing α6-integrin–Src–Akt signaling. In support of our hypothesis, TamR cells and associated mammospheres were found to exhibit elevated α6-integrin expression compared with their tamoxifen-sensitive counterparts. Furthermore, tumor sections from patients who relapsed on tamoxifen showed enhanced α6-integrin expression. Gene expression profiling from the TCGA database further revealed that basal-like breast cancer samples, known to be largely unresponsive to tamoxifen, demonstrated higher α6-integrin levels than luminal breast cancer samples. Importantly, AF reduced TamR cell viability and disrupted TamR mammospheres while concomitantly reducing α6-integrin messenger RNA and protein levels. In addition, AF and small interfering RNA against α6-integrin blocked tamoxifen-stimulated proliferation of TamR MCF-7 cells and further sensitized these cells to tamoxifen. Moreover, AF reduced Src and Akt signaling activation in TamR MCF-7 cells. Our findings suggest elevated α6-integrin expression is associated with tamoxifen resistance and AF suppresses α6-integrin–Src–Akt signaling activation to confer activity against TamR breast cancer. Fil: Campbell, Petreena S.. Loma Linda University School of Medicine; Estados Unidos Fil: Mavingire, Nicole. Loma Linda University School of Medicine; Estados Unidos Fil: Khan, Salma. Loma Linda University School of Medicine; Estados Unidos Fil: Rowland, Leah K.. Loma Linda University School of Medicine; Estados Unidos Fil: Wooten, Jonathan V.. Loma Linda University School of Medicine; Estados Unidos Fil: Opoku Agyeman, Anna. Loma Linda University School of Medicine; Estados Unidos Fil: Guevara, Ashley. Loma Linda University School of Medicine; Estados Unidos Fil: Soto, Ubaldo. Loma Linda University School of Medicine; Estados Unidos Fil: Cavalli, Fiorella. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina Fil: Loaiza Perez, Andrea Irene. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Nagaraj, Gayathri. Loma Linda University School of Medicine; Estados Unidos Fil: Denham, Laura J.. Loma Linda University School of Medicine; Estados Unidos Fil: Adeoye, Olayemi. Loma Linda University School of Medicine; Estados Unidos Fil: Jenkins, Brittany D.. Henry Ford Cancer Institute; Estados Unidos Fil: Davis, Melissa B.. Henry Ford Cancer Institute; Estados Unidos Fil: Schiff, Rachel. Baylor College of Medicine; Estados Unidos Fil: Brantley, Eileen J.. Loma Linda University School of Medicine; Estados Unidos

Because elevated glucocorticoid levels can impair reproduction, populations or species that engage in particularly valuable reproductive attempts may down-regulate the glucocorticoid stress response during reproduction (the brood value hypothesis). It is not clear, however, whether individuals rapidly modulate glucocorticoid responses based on shifting cues about the likelihood of reproductive success. By manipulating brood size to create broods that differed in potential value, we tested whether female barn swallows (Hirundo rustica) rapidly modulated the glucocorticoid stress response to promote investment in high-value broods, and whether nestling phenotype was influenced by treatment. Within-individual changes in female corticosterone, body mass, and measures of oxidative stress were unrelated to brood size treatment. Standard offspring provisioning rate did not differ across treatments; however, in the presence of a model predator, females raising enlarged broods maintained higher offspring feeding rates relative to control broods. Brood size did influence nestling phenotype. Nestlings from enlarged broods had lower body mass and higher baseline corticosterone than those from reduced broods. Finally, in adult females both baseline and stress-induced corticosterone were individually repeatable. Thus, while under moderately challenging environmental conditions brood size manipulations had context-dependent effects on parental investment, and influenced nestling phenotype, maternal glucocorticoid levels were not modulated based on brood value but were individually consistent features of phenotype during breeding.

Circulating tumor cells (CTCs) have significant implications in both basic cancer research and clinical applications. To address the limited availability of viable CTCs for fundamental and clinical investigations, effective separation of extremely rare CTCs from blood is critical. Ferrohydrodynamic cell separation (FCS), a label-free method that conducted cell sorting based on cell size difference in biocompatible ferrofluids, has thus far not been able to enrich low-concentration CTCs from cancer patients' blood because of technical challenges associated with processing clinical samples. In this study, we demonstrated the development of a laminar-flow microfluidic FCS device that was capable of enriching rare CTCs from patients' blood in a biocompatible manner with a high throughput (6 mL h−1) and a high rate of recovery (92.9%). Systematic optimization of the FCS devices through a validated analytical model was performed to determine optimal magnetic field and its gradient, ferrofluid properties, and cell throughput that could process clinically relevant amount of blood. We first validated the capability of the FCS devices by successfully separating low-concentration (∼100 cells per mL) cancer cells using six cultured cell lines from undiluted white blood cells (WBCs), with an average 92.9% cancer cell recovery rate and an average 11.7% purity of separated cancer cells, at a throughput of 6 mL per hour. Specifically, at ∼100 cancer cells per mL spike ratio, the recovery rates of cancer cells were 92.3 ± 3.6% (H1299 lung cancer), 88.3 ± 5.5% (A549 lung cancer), 93.7 ± 5.5% (H3122 lung cancer), 95.3 ± 6.0% (PC-3 prostate cancer), 94.7 ± 4.0% (MCF-7 breast cancer), and 93.0 ± 5.3% (HCC1806 breast cancer), and the corresponding purities of separated cancer cells were 11.1 ± 1.2% (H1299 lung cancer), 10.1 ± 1.7% (A549 lung cancer), 12.1 ± 2.1% (H3122 lung cancer), 12.8 ± 1.6% (PC-3 prostate cancer), 11.9 ± 1.8% (MCF-7 breast cancer), and 12.2 ± 1.6% (HCC1806 breast cancer). Biocompatibility study on H1299 cell line and HCC1806 cell line showed that separated cancer cells had excellent short-term viability, normal proliferation and unaffected key biomarker expressions. We then demonstrated the enrichment of CTCs in blood samples obtained from two patients with newly diagnosed advanced non-small cell lung cancer (NSCLC). While still at its early stage of development, FCS could become a complementary tool for CTC separation for its high recovery rate and excellent biocompatibility, as well as its potential for further optimization and integration with other separation methods.

Human intestinal organoids (HIOs) are millimeter-scale models of the human intestinal epithelium and hold tremendous potential for advancing fundamental and applied biomedical research. HIOs resemble the native gut in that they consist of a fluid-filled lumen surrounded by a polarized epithelium and associated mesenchyme; however, their topologically-closed, spherical shape prevents flow through the interior luminal space, making the system less physiological and leading to the buildup of cellular and metabolic waste. These factors ultimately limit experimentation inside the HIOs. Here, we present a millifluidic device called the gut organoid flow chip (GOFlowChip), which we use to "port" HIOs and establish steady-state liquid flow through the lumen for multiple days. This long-term flow is enabled by the use of laser-cut silicone gaskets, which allow liquid in the device to be slightly pressurized, suppressing bubble formation. To demonstrate the utility of the device, we establish separate luminal and extraluminal flow and use luminal flow to remove accumulated waste. This represents the first demonstration of established liquid flow through the luminal space of a gastrointestinal organoid over physiologically relevant time scales. Flow cytometry results reveal that HIO cell viability is unaffected by long-term porting and luminal flow. We expect the real-time, long-term control over luminal and extraluminal contents provided by the GOFlowChip will enable a wide variety of studies including intestinal secretion, absorption, transport, and co-culture with intestinal microorganisms.

Restitution of wounds in colonic epithelium is essential in the maintenance of health. Microbial products, such as the short-chain fatty acid butyrate, can have positive effects on wound healing. We used an in vitro model of T84 colonic epithelial cells to determine if the Snail genes Slug ( SNAI2) and Snail ( SNAI1), implemented in keratinocyte monolayer healing, are involved in butyrate-enhanced colonic epithelial wound healing. Using shRNA-mediated Slug/Snail knockdown, we found that knockdown of Slug (Slug-KD), but not Snail (Snail-KD), impairs wound healing in scratch assays with and without butyrate. Slug and Snail had differential effects on T84 monolayer barrier integrity, measured by transepithelial resistance, as Snail-KD impaired the barrier (with or without butyrate), whereas Slug-KD enhanced the barrier, again with or without butyrate. Targeted transcriptional analysis demonstrated differential expression of several tight junction genes, as well as focal adhesion genes. This included altered regulation of Annexin A2 and ITGB1 in Slug-KD, which was reflected in confocal microscopy, showing increased accumulation of B1-integrin protein in Slug-KD cells, which was previously shown to impair wound healing. Transcriptional analysis also indicated altered expression of genes associated with epithelial terminal differentiation, such that Slug-KD cells skewed toward overexpression of secretory cell pathway-associated genes. This included trefoil factors TFF1 and TFF3, which were expressed at lower than control levels in Snail-KD cells. Since TFFs can enhance the barrier in epithelial cells, this points to a potential mechanism of differential modulation by Snail genes. Although Snail genes are crucial in epithelial wound restitution, butyrate responses are mediated by other pathways as well.NEW & NOTEWORTHY Although butyrate can promote colonic mucosal healing, not all of its downstream pathways are understood. We show that the Snail genes Snail and Slug are mediators of butyrate responses. Furthermore, these genes, and Slug in particular, are necessary for efficient restitution of wounds and barriers in T84 epithelial cells even in the absence of butyrate. These effects are achieved in part through effects on regulation of β1 integrin and cellular differentiation state.

Isolation of circulating tumor cells (CTCs) from blood provides a minimally-invasive alternative into the basic understanding, diagnosis and prognosis of metastatic cancers. The roles and clinical values of CTCs are under intensive investigation, yet most studies are limited by technical challenges in the comprehensive enrichment of intact and viable CTCs with minimal white blood cells (WBCs) contamination. Here, we report a novel method based on contrast of cell magnetization in biocompatible ferrofluids (a colloidal magnetic nanoparticles suspension), termed as integrated ferrohydrodynamic cell separation (iFCS), that enriches CTCs in a tumor antigen-independent and cell size variation-inclusive manner, and achieves high-throughput (12 mL h(−1)), high recovery rate (99.08% at down to ~;10 cells mL(−1) spike ratio), low WBCs contamination (533 cells for every one milliliter blood processed) and is biocompatible. This method will enable large cohort research to define clinical and diagnostic value of CTC subtypes.

The Duffy Antigen Receptor for Chemokines (DARC/ACKR1) is an atypical chemokine receptor which promiscuously binds chemokines of both CC and CXC chemokine families. DARC/ACKR1 maintains homeostatic levels of chemokines in circulation through its expression on erythrocytes, and in tissues participates in chemokine transport, where chemokines are transported across the cell layer to help establish chemokine gradients. Establishment of these chemokine gradients is essential for proper immune cell trafficking. In our recent study, we have shown that DARC/ACKR1 is expressed on the tumor epithelium, and through in silico analysis of data from the Cancer Genome Atlas (TCGA), that higher DARC/ACKR1 expression is positively and significantly correlated with tumor-associated leukocyte abundance when using CIBERSORT deconvolution methods. Specifically, higher DARC/ACKR1 expression was linked with increases in B cell, T cell, monocyte and macrophage populations. Higher DARC/ACKR1 expression is also significantly associated with better survival outcomes, across all breast cancer molecular subtypes. To further evaluate DARC/ACKR1 expression and influences on the breast TME, we have employed multiple approaches to characterize how DARC/ACKR1 impacts immune cell recruitment and subsequent infiltration into the tumor. In our patient cohort, we have recently completed Hyperion imaging mass cytometry staining of tumors that have been scored by IHC as DARC/ACKR1 high or low expressing. We used a panel of approximately 30 markers, including structural, immune, and other markers of interest. Following imaging, we have used cell segmentation software to quantify marker expression on a cell-by-cell basis. Using this method, we are able to conduct single-cell analyses of our markers of interest, while also retaining the spatial composition of the cells. We have used the tSNE algorithm to identify cell populations that cluster uniquely within DARC/ACKR1 high or low tumor expressing groups, specifically identifying different clusters of immune cell populations, which show different spatial expression patterns in our comparison groups. In addition to investigations of immune cell recruitment to the TME, we have also developed a novel DARC/ACKR1 breast cancer transgenic mouse model to study how disease progression may differ with differing DARC/ACKR1 phenotypes. Briefly, DARC -/- female mice were crossed with the male C3(1)Tag +/0 breast cancer transgenic mouse to generate DARC +/-, C3(1)Tag +/0 male mice. These males were subsequently backcrossed to DARC -/- females, to generate the target DARC/ACKR1 +/- or -/-, C3(1)Tag+ mice. These mice show evidence of early disease beginning at 3.5 months of age, and progress to advanced disease by 5 months of age. We have collected tumor tissue and blood specimens from mice ranging from 3.5 to 7 months of age, to characterize disease progression over time between our DARC/ACKR1 expressing and non-expressing groups. We have observed differences in tumors characteristics between our DARC/ACKR1 expressing groups, and upon histological and immunofluorescent evaluation, we have begun to observe differences in immune cell recruitment, following similar patterns to what we observed in our in silico and patient cohort analyses. Citation Format: Rachel Martini, Brittany Jenkins, Dorrah Deeb, Hiranmayi Ravichandran, Paula Ginter, Esther Cheng, Syed Hoda, Dhananjay Chitale, Haythem Ali, Eleanor Walker, Jessica Bensenhaver, Olivier Elemento, Nancy Manley, Lisa Newman, Melissa Davis. Breast tumor expression of DARC/ACKR1 and impacts on disease progression and immune cell recruitment in the tumor microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4975.

Part of the delicate interplay of interactions between tumor cells, immune cells, and other cell types cells contribute to the complexities of the tumor microenvironment (TME). Specific interactions between chemokines and their receptors can influence the migration of immune cells, thereby directing tumor immune responses. In general immune response, the Atypical Chemokine Receptor 1 (ACKR1/DARC) modulates chemokine levels through the sequestration of proinflammatory chemokines in circulation and transcytosis across endothelial and epithelial tissue. This can affect not only the concentration of immune cells brought to the TME, but also the profile of immune cell types, given that ACKR1 is a promiscuous binder of both chemokine classes (CXCL and CCL). ACKR1 also serves as an ancestral informative marker. The gene harbors a mutation (“Duffy-null”) in its promoter/5′ UTR region that abolishes its normal erythrocytic expression, a status that only exists in populations of sub-Saharan African descent, conferring an evolutionary advantage against malaria. This polymorphism is carried by 60-80% of African-Americans, and based on our findings, it can influence chemokine and immune cell migration in the context of tumorigenesis. Our study reveals the potential effect of this mutation in women with breast cancer (BC) by showing correlations between circulating chemokine concentrations, measured through Luminex multiplexing immunoassays, and relative levels of ACKR1 and tumor-infiltrating lymphocytes (TIL), scored through immunohistochemistry. Our initial investigation of epithelial expression of ACKR1 on breast tumors revealed supporting data of our hypothesis that differential expression of ACKR1 on breast tumor tissue is correlated with a distinct signature of TILs and associated proinflammatory chemokines. Specifically, lower levels of tumor-associated ACKR1 were seen in African-Americans, in addition to a less robust signature of TILs, including macrophages and T-cells. Genotyping for the Duffy-null mutation also showed correlations with proinflammatory chemokines, including CCL2 and CXCL8. Our data suggest that ACKR1 levels in circulation and tissues can indirectly influence the immune cell profile in African-American women with BC, potentially leading to changes in tumor aggressiveness and response to treatment. Citation Format: Brittany D. Jenkins, Talina Fleifel, Rachel N. Martini, Haythem Ali, Lisa A. Newman, Melissa B. Davis. Tumor-associated ACKR1/DARC is correlated with a unique signature of proinflammatory chemokines and TILs in African-American women with breast cancer [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B056.

In clinic, we observe that women of African descent have higher incidence rates of breast cancer (BC). These women are disproportionately diagnosed with the most aggressive subtype of BC, triple-negative BC, compared to women belonging to any other ancestry groups. To investigate factors driving this disparity, our work focuses on the tumor microenvironment (TME), specifically through the lens of an atypical chemokine receptor. The Duffy Antigen Receptor for Chemokines (DARC) is a nonsignaling, atypical chemokine receptor that binds two structural classes of chemokines. DARC modulates chemokine availability in circulation and participates in chemokine transport in tissues, to recruit immune cells back to sites of inflammation. DARC additionally serves as a portal of entry for the malaria-causing parasite Plasmodium vivax. In Sub-Saharan Africa, where malaria is endemic, a regulatory variant arose removing DARC expression from red blood cells (RBCs). This mutation, known as the Duffy-Null allele, was highly beneficial in this population, and quickly rose to fixation. Despite being called Duffy-Null, these individuals retain DARC expression in other cell types, only lacking expression on RBCs. Knowing that the Duffy-Null allele is prevalent in this population of women, we aim to investigate how DARC tumor expression and Duffy-Null status impacts the TME in silico through analysis of The Cancer Genome Atlas (TCGA) data, and in vivo through establishment of mouse models that depict Duffy-Null status in the BC TME. Using TCGA data, we see that DARC tumor gene expression is significantly lower among African-Americans compared to Whites. Using the CIBERSORT online platform, we quantified tumor-associated leukocyte (TAL) abundance in the TME. Overall, we found that tumors with high DARC expression have significantly higher total TAL abundance (p < 0.0001), and that DARC expression and TAL abundance are positively and significantly correlated (R = 0.545, p < 0.0001). Specifically, DARC expression influenced levels of B cell, T cell, monocyte and macrophage population in the TME. In our mouse models, we have utilized the C3(1)Tag BC transgenic mouse model, alongside a DARC knock-out mouse. To better recapitulate the Duffy-Null phenotype in our mouse model, our approach is to generate a BC/Duffy-Null bone marrow derived (BMD) chimera, where a DARC+/-; C3(1)Tag+/0 recipient will receive bone marrow from a DARC -/- donor. The resulting chimera mice will retain DARC expression in all cells, aside from bone marrow-derived cells from the null donor. In the C3(1)Tag line, disease onset is at approximately 12 weeks, at which time we will assess changes in disease progression in our chimeras compared to controls by collecting total number of tumors, tumor location, tumor size and weight, and hematoxylin and eosin staining of tumor sections, and immune cell infiltration quantification. Citation Format: Rachel Martini, Brittany Jenkins, Clayton Yates, Lisa Newman, Nancy Manley, Melissa Davis. In silico and in vivo analysis of the Duffy Antigen Receptor for Chemokines (DARC) in the breast tumor microenvironment [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B058.

Disparities in breast cancer survival among ethnic groups have been a persistent finding over the past five decades, exacerbated in part by the lack of improvement to non-white patient outcomes, despite treatment advancements that have improved clinical outcomes in white women. A significant part of this disparity is health equity; however, recent evidence from several groups indicates that histologic and pathologic diversity in tumor phenotypes among ethnic groups is also a key factor affecting the differences in clinical outcome. Specifically, correlated findings among women with significant West African ancestry reveal that there is a genetic link between women across the African Diaspora that is associated with aggressive tumor phenotypes, including triple-negative breast cancer. Aside from the global incidence of TNBC being higher in regions with relatively higher numbers of women with African ancestry, we also find that pathologic progression of tumors in African Americans tends to mimic that of African women. Tumor progression is directly related to the immune response elicited by the onset of tumor growth as well as the underlying tissue microenvironment, particularly the inflammatory status. We have identified several lines of evidence that suggest there is a distinct immune response to breast cancer, which is also tumor phenotype/subtype specific, when comparing patients of significant African ancestry with those of primarily European ancestry. These findings suggest that there could be a unique mechanism of tumor immunology at work, driven by population private genetic alleles derived in Africa and transmitted throughout the African Diaspora, causing a unique tumor phenotype in these breast cancer patients. This unique phenotype is likely the key factor in distinct treatment responses that result in poorer clinical outcomes for African American women. Citation Format: Melissa B. Davis, Brittany D. Jenkins, Rachel A. Martini, Haythem Ali, Clayton C. Yates, Elizabeth A. Howerth, Petros Nikolinakos, Michele Monteil, Lisa A. Newman. Exploring the impact of African ancestry in tumor immune response, a possible role in disparate clinical outcomes [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr IA27.

Tumor-specific immune response is an important aspect of disease prognosis and ultimately impacts treatment decisions for innovative immunotherapies. The Atypical Chemokine Receptor 1 (ACKR1/DARC) gene, plays a pivotal role in immune regulation and harbors several Single Nucleotide Variants (SNVs) that are specific to Sub-Saharan African Ancestry. In this study, we report the clinical relevance of DARC/ACKR1 tumor expression in breast cancer, in the context of a tumor immune response that may be associated with Sub-Saharan African Ancestry. We found that in infiltrating carcinomas from The Cancer Genome Atlas (TCGA) breast cohort (n=838), African-Americans (AA) have a higher proportion of tumors with low DARC/ACKR1- expression when compared to White Americans (WA), and DARC/ACKR1 tumor expression is positively correlated with pro-inflammatory chemokine, CCL2/MCP-1 (p

Interactions between the gut microbiota and the host are important for health, where dysbiosis has emerged as a likely component of mucosal disease. The specific constituents of the microbiota that contribute to mucosal disease are not well defined. The authors sought to define microbial components that regulate homeostasis within the intestinal mucosa. Using an unbiased, metabolomic profiling approach, a selective depletion of indole and indole-derived metabolites was identified in murine and human colitis. Indole-3-propionic acid (IPA) was selectively diminished in circulating serum from human subjects with active colitis, and IPA served as a biomarker of disease remission. Administration of indole metabolites showed prominent induction of IL-10R1 on cultured intestinal epithelia that was explained by activation of the aryl hydrocarbon receptor. Colonization of germ-free mice with wild-type Escherichia coli, but not E. coli mutants unable to generate indole, induced colonic epithelial IL-10R1. Moreover, oral administration of IPA significantly ameliorated disease in a chemically induced murine colitis model. This work defines a novel role of indole metabolites in anti-inflammatory pathways mediated by epithelial IL-10 signaling and identifies possible avenues for utilizing indoles as novel therapeutics in mucosal disease.

Phenotypic differences among individuals are often linked to differential survival and mating success. Quantifying the relative influence of genetic and environmental variation on phenotype allows evolutionary biologists to make predictions about the potential for a given trait to respond to selection and various aspects of environmental variation. In particular, the environment individuals experience during early development can have lasting effects on phenotype later in life. Here, we used a natural full-sib/half-sib design as well as within-individual longitudinal analyses to examine genetic and various environmental influences on plumage color. We find that variation in melanin-based plumage color – a trait known to influence mating success in adult North American barn swallows (Hirundo rustica erythrogaster) – is influenced by both genetics and aspects of the developmental environment, including variation due to the maternal phenotype and the nest environment. Within individuals, nestling color is predictive of adult color. Accordingly, these early environmental influences are relevant to the sexually selected plumage color variation in adults. Early environmental conditions appear to have important lifelong implications for individual reproductive performance through sexual signal development in barn swallows. Our results indicate that feather color variation conveys information about developmental conditions and maternal care alleles to potential mates in North American barn swallows. Melanin-based colors are used for sexual signaling in many organisms, and our study suggests that these signals may be more sensitive to environmental variation than previously thought.

A fundamental element of how vertebrates respond to stressors is by rapidly elevating circulating glucocorticoid hormones. Individual variation in the magnitude of the glucocorticoid stress response has been linked with reproductive success and survival. But while the adaptive value of this response is believed to stem in part from changes in the expression of hormone-mediated behaviors, it is not clear how the behavior of stronger and weaker glucocorticoid responders differs during reproduction, or during exposure to ecologically relevant stressors. Here we report that in a population of barn swallows (Hirundo rustica erythrogaster) experiencing high rates of nest predation, circulating levels of corticosterone (the primary avian glucocorticoid) during exposure to a standardized stressor predict aspects of subsequent behavior and fitness. Individuals that mounted a stronger corticosterone stress response during the early reproductive period did not differ in clutch size, but fledged fewer offspring. Parents with higher stress-induced corticosterone during the early reproductive period later provisioned their nestlings at lower rates. Additionally, in the presence of a model predator stress-induced corticosterone was positively associated with the latency to return to the nest, but only among birds that were observed to return. Model comparisons revealed that stress-induced hormones were better predictors of the behavioral and fitness effects of exposure to transient, ecologically relevant stressors than baseline corticosterone. These findings are consistent with functional links between individual variation in the hormonal and behavioral response to stressors. If such links occur, then selection on the heritable components of the corticosterone stress response could promote adaptation to novel environments or predation regimes.

Life history theory describes the optimization of important trade-offs within an individual’s lifetime and predicts that an individual’s reproductive performance (RP) will improve up until a point of senescence. Despite abundant evidence for this pattern, relatively few studies consider the mechanisms associated with age-related improvements in RP. In this study, we aimed to describe patterns of age-related RP (seasonal fledgling production) in female North American barn swallows (Hirundo rustica erythrogaster) using a longitudinal data set to test multiple hypotheses about the social, morphological, and ecological factors underlying this prominent life history pattern. To address these objectives, we used generalized linear mixed models in a three-step series of analyses in which we assessed (1) patterns of female age-related RP; (2) the influence of age on changes in social, morphological, and ecological factors; and (3) whether the changes in RP were concomitant with changes in these factors. We found that (1) females showed patterns of age-related reproduction, in which performance increased in the first 2 years of breeding and decreased thereafter, (2) female tail streamer length increased and the extent of breast coloration increased then decreased significantly with age, and (3) changes in morphological traits did not covary with changes in reproductive performance over time. Our within-individual results highlight the importance of considering explicit links between morphology and reproductive performance that are not easily captured by population-level analyses.

Breast cancer (BC) is a heterogeneous disease that leads to varied molecular subtypes and distinct clinical outcomes. Tumor heterogeneity, which is in part influenced by genetic ancestry, contributes to racial disparities in BC. This disparity has persisted for over 30 years, despite improvements in detection, screening, and treatment methods. When investigating race-specific differences in the breast tumor microenvironment (TME), we believe that chemokine receptors, such as atypical chemokine receptor 1 (ACKR1/DARC), play an integral role in regulating chemokine and immune cell migration in circulation and the TME, ultimately influencing tumor progression. ACKR1/DARC specifically plays a role in ancestry-related differences in BC due to an African-specific ancestral allele (“Duffy-null” mutation) that is fixed in Sub-Saharan African populations, and present in 60-70% of African-Americans. Those that harbor the mutation do not express ACKR1/DARC on erythrocytes, ultimately affecting immune cell migration in these populations only. To better understand the effects of this variant in circulation and the TME, we performed immunohistochemistry (IHC) on breast tumors from a cohort of patients with matched blood samples. From our IHC analysis, we quantified levels of ACKR1/DARC, in addition to levels of target pro-inflammatory chemokines, and distinct immune populations of T-cells and macrophages. In addition, individuals were genotyped to determine Duffy-null status, and a correlative Luminex multiplex assay was performed on patient plasma to determine concentrations of pro-inflammatory chemokines in circulation. We determined that those with low expression of ACKR1/DARC in tumors exhibited a unique signature of immune cell infiltrates that would encourage a more aggressive TME. In addition, we observed variable levels of circulating chemokines, where those with the Duffy-null mutation exhibited significantly decreased levels of CCL2. Overall, our analyses support the hypothesis that tumor-specific DARC/ACKR1 plays a vital role in immune cell regulation in women with BC. Citation Format: Brittany D. Jenkins, Rachel Martini, Kevin Gardner, Michele Monteil, Dorrah Deeb, Lisa Newman, Melissa Davis. The functional role of atypical chemokine receptor 1 in immune cell regulation of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4565.

Introduction: Exosomes are communication vesicles between tumor cells and immune cells. However, the mechanism underlining this cell-cell communication is not well understanding, particularly in African American (AA) breast cancer patients. Kaiso, a bi-modal transcription factor is highly expressed in AA patients and high Kaiso expression correlates with aggressiveness and the disparity in survival outcomes compared to European American (EA) patients. However, the biological consequences of Kaiso in immune signaling of breast cancer exosomes has not been studied. Herein we demonstrate the biological role of Kaiso in immune signaling in breast cancer exosomes. Methods: We utilized Nanostring immune profiling technology along with multiple in vitro and in vivo assays to study the role of Kaiso in breast cancer immune escape. Results: Nanostring pan cancer immune profiling showed that EA breast cancer exosomes exhibited higher expression of TILs markers, T cell activation markers and CD8+T Cells markers compared to AA, while we observed an increase in the expression of anti-phagocytic molecule CD47 in breast cancer patient exosomes of AA compared to EA. In addition to that CD47 and SIRP-α (Signal Regulatory Protein) are highly expressed in Kaiso-scrambled MDA-MB-231 cells (sh-SCR) and exosomes, whereas THBS1, which is a regulator of CD47 expression and is regarded as angiogenesis inhibitor is significantly increased in sh-Kaiso MDA-231 cells and exosomes. Additionally, we observed that Kaiso directly binds methylated sequences in the promoter region of CD47 and THBS1 by ChIP assay. Furthermore, in vivo sh-Kaiso cells injected into athymic mice exhibited delayed tumor formation after four weeks with smaller tumor size as compared to sh-SCR cells, and we observed higher expression of THBS1 with lower expression of CD47 and SIRP-α molecules by IHC and exosomes isolated from invivo tumors, indicating that Kaiso is associated with macrophage mediated immune escape. Conclusion: Our findings demonstrate the role of kaiso in immune signaling through exosomes which may be related with more aggressive cancer phenotype in breast cancer specially in African Americans. Citation Format: Md Shakir U. Ahmed, Brittany Jenkins, Benjamin Adu-Addai, Balasubramanyam Karanam, Melissa B. Davis, William E. Grizzle, Honghe Wang, Clayton C. Yates. Kaiso influences immune signaling of breast cancer exosomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1525.

In studies of the tumor microenvironment (TME), factors that influence immune cell infiltration are of great interest, as these populations can influence disease prognosis, and potential treatment for patients. Through in silico analysis of TCGA breast cancer (BC) cohort data, we have identified the Duffy antigen receptor for chemokine/atypical chemokine receptor 1 (DARC/ACKR1) as a potential driver of immune cell infiltration in the BC TME. DARC/ACKR1 is an atypical chemokine receptor that modulates levels of chemokine both in circulation through expression on red blood cells, and participates in chemokine transcytosis in tissues through expression on both post-capillary venules of endothelial cells and epithelial cells. As chemokines establish gradients to recruit specific immune cell types, we hypothesize that the function of DARC/ACKR1 in chemokine level modulation also influences tumor-associated immune cell levels. In the TCGA BC cohort, we observe a strongly positive and significant correlation between DARC/ACKR1 expression and total number of tumor-associated leukocytes, specifically populations of B cells, T cell, monocytes and macrophages. To further characterize the role of DARC/ACKR1 in vivo, we have established a novel DARC knock-out BC transgenic mouse model to study DARC/ACKR1 in the BC TME. To develop our target experimental mice, Ackr1-/- female mice were crossed with Ackr1 +/-; C3(1)Tag +/0 to generate the target Ackr1 -/-; C3(1)Tag +/0 experimental mice. Experimental mice and the littermate controls were aged to 3.5 months, at which point they were euthanized. Blood was collected from the mice to characterize the circulating chemokine profile. Mammary glands containing tumors were fixed and paraffin embedded, followed by IHC and immunofluorescent staining for expression of DARC/ACKR1, target chemokines, immune cells, epithelial cells and endothelial cells. The remaining glands were dissociated, followed by fluorescent-activating cell sorting analysis (FACS) to quantify abundance of immune cells in the gland. Using these methods, we have defined the circulating chemokine profile alongside patterns of immune cell infiltration in our target DARC/ACKR1 knockout BC mice compared to littermate controls in mouse mammary tumors. Citation Format: Rachel N. Martini, Brittany D. Jenkins, Lisa A. Newman, Nancy Manley, Melissa B. Davis. In vivo characterization of the Duffy antigen receptor for chemokines (DARC/ACKR1) in breast cancer tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4630.

Tumor-associated immune cells, stroma and several other cell types make up the complex tumor microenvironment (TME) that contributes to a broad spectrum of potential clinical outcomes for breast cancer (BC) patients. Part of this delicate interplay is the interaction between pro-inflammatory chemokines and their receptors, which direct the migration of immune cells to areas of tumor-associated inflammation. Our focus in this complex process is on the role of the Atypical Chemokine Receptor 1 (ACKR1/DARC). In general immune response, its expression on erythrocytes helps to maintain chemokine homeostasis by sequestering chemokines in circulation while its expression on endothelial tissue transcytoses the chemokines from tissue into circulation, which ultimately affects which immune cells are brought to the TME. Its role in epithelial tissue expression is less understood. The purpose of this study is to investigate differential gene expression of ACKR1 in breast epithelial tumor tissue through IHC methods, and to determine how that expression correlates with both circulating and infiltrating proinflammatory chemokines. In addition, we will show this role to be associated with specific classes of tumor-infiltrating lymphocytes (TIL) in BC. Circulating chemokine levels for a variety of ACKR1-associated proinflammatory chemokines were determined using Luminex multiplexing assays. Results from our study cohort indicate differential expression of ACKR1 on epithelial tumor tissue, which correlated with a unique signature of immune cell infiltrates and associated proinflammatory chemokines. Tumors positive for AKCR1 expressed higher levels of circulating and infiltrating CCL2 (MCP-1) and lower levels of CXCL8 (IL-8). Tumor-expressing ACKR1 was also found to be associated with T-cells, B-cells, macrophages, and monocytes, where positive tumors tended to express a more robust profile of TILs. Our preliminary data suggest the presence or absence of ACKR1 on breast epithelial tumor tissue can influence the chemokine and immune cell profile within the TME, which can ultimately influence tumor aggressiveness, proliferation, and response to treatments, such as immunotherapies. Citation Format: Brittany D. Jenkins, Talina Fleifel, Rachel Martini, Haythem Ali, Lisa Newman, Melissa Davis. Tumors expressing ACKR1 exhibit a unique signature of tumor-infiltrating immune cells in women with breast cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A081.

The regulation of immune cell infiltration into the tumor microenvironment can influence disease prognosis. The specific populations that are present can inform potential treatment options. This work investigates immune cell regulation in the breast cancer tumor microenvironment, specifically how an atypical chemokine receptor, known as the Duffy Antigen Receptor for Chemokines (DARC/ACKR1) can influence levels of leukocyte populations in the breast tumor environment. DARC is a nonsignaling receptor able to bind both the CC and CXC classes of chemokines, and mainly functions to modulate levels of chemokines in circulation, and aid in chemokine transport in tissues. In this regard, DARC expression may determine the profile of immune response.To investigate DARC expression and its effects on tumor-associated leukocyte (TAL) populations, we obtained RNA-seq data from The Cancer Genome Atlas (TCGA) Breast Cancer cohort. We proceeded through our analysis with those samples denoted as primary tumor samples (n=400). To estimate the relative abundance of TAL populations, we used the CIBERSORT online platform, which estimates fractions of 22 different TAL populations based on gene expression data. After completing the CIBERSORT analysis, we proceeded with only those samples that had a significant CIBERSORT output (n=167). In our analysis, we found that the total abundance of all TALs was significantly higher in tumors that have high DARC expression (p < 0.0001), and that DARC expression and the TAL abundance are positively and significantly correlated (R = 0.545, p < 0.0001). When we investigated specific TAL populations, we saw that B cell, T-cell, monocyte and macrophage populations were significantly increased in tumors with high DARC expression. Citation Format: Rachel Martini, Brittany D. Jenkins, Clayton Yates, Lisa Newman, Melissa Davis. The Duffy Antigen Receptor for Chemokines (DARC) influences levels of tumor-associated leukocytes in the breast tumor microenvironment [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A090.

Sexually selected traits confer greater reproductive benefits to individuals with more elaborate forms of the signal. However, whether these signals convey reliable information about the physiology underlying trait development remains unknown in many species. The steroid hormone corticosterone (CORT) mediates important physiological and behavioral processes during the vertebrate stress response, and CORT secretion itself can be modulated by melanocortins. Thus, sexually selected melanin-based plumage coloration could function as an honest signal of an individual's ability to respond to stressors. This hypothesis was tested in North American barn swallows, Hirundo rustica erythrogaster, where males with darker ventral plumage color exhibit higher phaeomelanin content and are more successful at reproduction. Because reproductive behavior occurs months after plumage signals are developed, we also addressed the potential temporal disconnect of physiological state during trait development and trait advertisement by analyzing three different measurements of CORT levels in adult males during the breeding season (trait advertisement) and in nestling males while they were growing their feathers (trait development). Variation in adult plumage color did not predict baseline or stress-induced CORT, or stress responsiveness. Likewise, there was no relationship between nestling plumage color and any of the CORT measurements, but heavier nestlings had significantly lower baseline CORT. Our finding that a predominantly phaeomelanin-based trait is unrelated to circulating CORT suggests that phaeomelanin and eumelanin signals may convey different physiological information, and highlights the need for further study on the biochemical links between the hypothalamic-pituitary-adrenal (HPA) axis and the production of different melanin-based pigments.

Shih-Fan, Chan/0000-0003-2955-9433; Shen, Sheng-Feng/0000-0002-0631-6343; Albrecht, Tomas/0000-0002-9213-0034; Karaardic, Hakan/0000-0001-9839-4201; Wilkins, Matthew/0000-0002-3162-6749 WOS: 000381578200007 PubMed: 27357267 Population divergence in geographic isolation is due to a combination of factors. Natural and sexual selection may be important in shaping patterns of population differentiation, a pattern referred to as 'isolation by adaptation' (IBA). IBA can be complementary to the well-known pattern of 'isolation by distance' (IBD), in which the divergence of closely related populations (via any evolutionary process) is associated with geographic isolation. The barn swallow Hirundo rustica complex comprises six closely related subspecies, where divergent sexual selection is associated with phenotypic differentiation among allopatric populations. To investigate the relative contributions of selection and geographic distance to genome-wide differentiation, we compared genotypic and phenotypic variation from 350 barn swallows sampled across eight populations (28 pairwise comparisons) from four different subspecies. We report a draft whole-genome sequence for H. rustica, to which we aligned a set of 9493 single nucleotide polymorphisms (SNPs). Using statistical approaches to control for spatial autocorrelation of phenotypic variables and geographic distance, we find that divergence in traits related to migratory behaviour and sexual signalling, as well as geographic distance, together explain over 70% of genome-wide divergence among populations. Controlling for IBD, we find 42% of genomewide divergence is attributable to IBA through pairwise differences in traits related to migratory behaviour and sexual signalling alone. By (i) combining these results with prior studies of how selection shapes morphological differentiation and (ii) accounting for spatial autocorrelation, we infer that morphological adaptation plays a large role in shaping population -level differentiation in this group of closely related populations. National Science FoundationNational Science Foundation (NSF) [DEB-CAREER-1149942]; NSFNational Science Foundation (NSF); Royal Society of LondonRoyal Society of London; Ministry of Education, Youth and Sports of the Czech RepublicMinistry of Education, Youth & Sports - Czech Republic [LH14045] We thank C. Alex Buerkle for collaboration on all aspects of this project and the Safran, Kane and Flaxman laboratory groups at the University of Colorado for feedback and advice. We thanks Matthew Evans for collaboration on barn swallows in the UK. Funding for this project was provided by the National Science Foundation (DEB-CAREER-1149942) to R.J.S. M.R.W was supported by an NSF graduate research fellowship and P.N by a Royal Society of London University Research Fellowship. Work in the Czech Republic was funded by the Ministry of Education, Youth and Sports of the Czech Republic (project LH14045). All field and laboratory protocols were compliant with University of Colorado, and Federal animal care and import/export protocols. We are grateful for the excellent reviews provided by Dr. Anna Santure and three anonymous reviewers.

PREMISE OF THE STUDY Population genetic analyses provide information on the population context in which evolutionary processes operate and are important for understanding the evolution of geographically variable traits. Earlier studies showed that cone structure of lodgepole pine in the Rocky Mountains diverged among populations because of geographic variation in coevolutionary interactions involving mammalian and avian seed predators. Analyses of population genetic variation are needed to determine whether this divergence has arisen despite extensive gene flow and whether populations to the east and west of the Rocky Mountains have evolved convergent phenotypes independently. METHODS We investigated genetic structuring across 22 stands of lodgepole pine in the central Rocky Mountains and in isolated peripheral populations that experience different seed predators and exhibit parallel divergence in cone traits using a set of nine simple sequence repeats and 235 AFLP loci. KEY RESULTS Our analyses reveal high levels of genetic diversity within and low genetic differentiation among populations. Nonetheless, geographic and genetic distances were correlated, and isolated populations to the east and west of the Rocky Mountains had higher levels of differentiation than did populations in the central part of the range. CONCLUSIONS These data indicate not only that adaptive divergence of cone traits across a geographic mosaic of coevolution has occurred despite minimal genetic differentiation, but also that isolated populations to the east and west of the Rocky Mountains have evolved distinctive cones independently and in parallel. The population structure quantified here will inform future research aimed at detecting genetic variants associated with divergent adaptive traits.

Chemokines and their receptors can interact to affect the host's anti-tumor response by influencing the migration of host immune cells indirectly via a chemokine gradient. The regulation and function of these receptors is paramount in ensuring an appropriate immune response to an inflammatory stimulus, especially in the case of cancer. One such receptor, Atypical Chemokine Receptor 1 (ACKR1/DARC), is a genetically diverse transmembrane GPCR that can buffer pro-inflammatory CXC- and CC- chemokine activity in circulation and within tissues, influencing leukocyte migration. While the basic immunological function of this receptor is known, the specific function of its two genetic isoforms, A and B, is not. These isoforms are produced by two alternative promoters and distinct mRNA splicing events. Isoform A is not split by introns, even in the untranslated regions, and offers homology to CXCL8. The alternative splice variant, Isoform B, does contain an intron, and follows the GT-AG splicing rule, including a novel exon coding for an additional 7 amino acids. This localizes to the N-terminus, making up part of the extracellular binding region of the receptor. We aim to determine if the two major ACKR1 isoforms have distinct functional abilities to bind or sequester pro-inflammatory chemokines in epithelial breast cell lines. By creating isoform specific overexpression and knockout constructs, and transiently transfecting the DNA into our subtype specific cell lines, we can use an ELISA assay to determine chemokine concentration in the media, and measure differences based on isoform specificity. Using immunofluorescence, we first show that ACKR1 and our two proinflammatory chemokines, CCL2 and CXCL8, are expressed on the surface of epithelial cells and colocalize. We then indicate that there is an isoform specific difference in chemokine concentration between different breast cancer subtypes, in addition to differences seen in overexpression and knockout cell lines. Determining the functionality of these two isoforms can help us better understand ACKR1's role in the immune system, and its potential role as a prognostic marker in breast cancer. Citation Format: Brittany D. Jenkins, Rachel N. Martini, Inasia Brown, Melissa B. Davis. The functional relevance of Atypical Chemokine Receptor 1 (ACKR1/DARC) genetic isoforms in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5071.

Previous studies have shown that sexual signals can rapidly diverge among closely related species. However, we lack experimental studies to demonstrate that differences in trait-associated reproductive performance maintain sexual trait differences between closely related populations, in support for a role of sexual selection in speciation. Populations of Northern Hemisphere distributed barn swallows Hirundo rustica are closely related, yet differ in two plumage-based traits: ventral color and length of the outermost tail feathers (streamers). Here we provide experimental evidence that manipulations of these traits result in different reproductive consequences in two subspecies of barn swallow: (H. r. erythrogaster in North America and H. r. transitiva in the East Mediterranean). Experimental results in Colorado, USA, demonstrate that males with (1) darkened ventral coloration and (2) shortened streamers gained paternity between two successive reproductive bouts. In contrast, exaggeration of both traits improved reproductive performance within H. r. transitiva in Israel: males with a combination treatment of darkened ventral coloration and elongated streamers gained paternity between two successive reproductive bouts. Collectively, these experimental results fill an important gap in our understanding for how divergent sexual selection maintains phenotype differentiation in closely related populations, an important aspect of the speciation process.

By studying the breast cancer microenvironment, we can specifically address novel aspects of cancer development and progression at the molecular level. Chemokines are small signaling molecules that are an important component of this microenvironment, as they shape cellular composition of this space through recruitment of immune cells, among other functions. DARC, the Duffy Antigen Receptor for Chemokines, is an atypical chemokine receptor (ACKR1) that can bind different classes of chemokines. It is a non-signaling receptor that mainly acts as a regulator of chemokine homeostasis by removing them from circulation to recruit appropriate immune cell types. The purpose of this study is to determine how population-private variants of the DARC/ACKR1 gene across global population render distinct function and define aspects of human diversity in immune responses. A well-studied example of this would be the Duffy-null allele. This allele carries a mutation that is population-specific to recent decedents of Sub-Saharan Africans, and removes expression of DARC on red blood cells. Loss of expression on red blood cells presumably causes these “Duffy-Null” individuals to lose the ability to sequester chemokines, therefore losing homeostatic balance of these molecules in circulation. To identify additional mutations, we completed in silico analysis of minor allele frequencies across the gene in the 1000 Genomes Project data, and identified several other putative functional mutations that likely result in additional gene variants, specific to geographic ancestry groups and likely under similar selection as the Duffy null allele. We have prioritized our initial functional study to include mutations in the regulatory regions of the gene, as these mutations can change how and where the gene is expressed. Future work is focused on investigating these functional variants in situ, using human tissue and human breast cell lines, by isolating and re-engineering ancestry-specific variants in these cell lines. By identifying and determining functional outcomes of these population-specific mutation, we can observe differences in immune responses across these global populations, further defining the role that DARC/ACKR1 plays in the breast tumor microenvironment. Citation Format: Rachel Martini, Brittany Jenkins, Melissa Davis. Mutations in the Duffy Antigen Receptor for Chemokines (DARC/ACKR1) gene result in population-private variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1283. doi:10.1158/1538-7445.AM2017-1283

Interactions between chemokines and their receptors can improve a host’s anti-tumor response by influencing the targeted migration of immune cells via a chemokine gradient. Atypical Chemokine Receptor 1 (ACKR1/DARC), a genetically diverse transmembrane GPCR, acts as a decoy receptor for a variety of CXC and CC chemokines, including those with pro-malignant and pro-inflammatory effects, such as CCL2 (MCP-1, MCAF, JE) and CXCL8 (IL-8). The purpose of this study is to determine if the migration of tumor-associated immune cells is unique based on epithelial ACKR1 expression on breast cancer cells, and if this association is correlated to an increase in pro-malignant chemokines, better survival odds, and differences in race. Immunohistochemistry techniques were used to determine expression levels of ACKR1 on primary breast tumors, along with relative expression of T-cells, B-cells, dendritic cells, and macrophages. Concentrations of pro-inflammatory chemokines in circulation were determined using a Luminex-based immunoassay and matched patient peripheral blood samples. In silco analyses were performed to determine associations between ACKR1 tumor expression status, race, and survival. Finally, using human breast cancer cell lines and immunofluorescence techniques, co-localization between ACKR1 and selected pro-inflammatory chemokines was investigated. Results from these tests indicate that there is differential expression of immune cell types in tumors expressing ACKR1, and this difference was associated with the migration of B-cells and dendritic cells, which were not detected in ACKR1 negative tumors. Significantly increased circulating CCL2 and CXCL8 chemokine levels we also determined to be positively correlated with ACKR1 expression in primary breast tumors. Survival analyses showed a significantly increased relapse free survival in patients having tumors with high ACKR1 expression, while investigations into racial differences revealed a significant race effect, with Caucasians having higher ACKR1 levels on their tumors than African-Americans. Finally, co-localization between ACKR1 with CCL2 and CXCL8 is observed in cultured human breast cancer cells. Given that the data collected shows a tendency for those tumors positively expressing ACKR1 to have a more favorable prognosis, we suggest that a partial role of ACKR1 on breast tumor cells is to sequester pro-inflammatory chemokines in the tumor microenvironment, indirectly recruiting a distinct subset of tumor-associated immune cells. Citation Format: Brittany D. Jenkins, Rupali Hire, Elizabeth Howerth, Michele Monteil, Rachel Martini, Melissa B. Davis. Atypical chemokine receptor 1 (ACKR1/DARC) expressing tumors are associated with distinct recruitment of immune cells and increased pro-inflammatory chemokines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 953. doi:10.1158/1538-7445.AM2017-953

e23072 Background: Interactions between chemokines and their receptors can regulate anti-tumor response by influencing the migration of immune cells. Atypical Chemokine Receptor 1 (ACKR1/DARC), a genetically diverse transmembrane GPCR, acts as a decoy receptor for a variety of CXC and CC chemokines, including those with pro-malignant and pro-inflammatory effects, such as CCL2 and CXCL8 . The purpose of this study is to determine if the migration of tumor-associated immune cells is unique based on epithelial ACKR1 expression on breast cancer cells, and if this association is correlated to an increase in pro-malignant chemokines, survival, or race. Methods: Immunohistochemistry techniques were used to determine expression of ACKR1 on primary breast tumors, along with T-cells, B-cells, dendritic cells, and macrophages. Concentrations of pro-inflammatory chemokines in circulation were determined using a Luminex-based immunoassay. In silco analyses were performed to determine associations between ACKR1 tumor status, race, and survival. Finally, using human breast cancer cell lines and immunofluorescence techniques, co-localization between ACKR1 and pro-inflammatory chemokines was investigated. Results: Results from these tests indicate that there is differential infiltration of immune cell types in tumors expressing ACKR1, , which were not detected in ACKR1 negative tumors. Significantly increased circulating CCL2 and CXCL8 chemokine levels we also determined to be positively correlated with ACKR1 expression in primary breast tumors. Survival analyses showed a significantly increased relapse free survival in patients having tumors with high ACKR1 expression, while investigations into racial differences revealed a significant race effect, with Caucasians having higher ACKR1 levels on their tumors than African-Americans. Finally, co-localization between ACKR1 with CCL2 and CXCL8 is observed in cultured human breast cancer cells. Conclusions: tumors positively expressing ACKR1 to have a more favorable prognosis suggest that a role of ACKR1 on breast tumor cells is to sequester pro-inflammatory chemokines in the tumor microenvironment, recruiting a distinct subset of tumor-associated immune cells.

The tumor microenvironment is a complex heterogeneous mixture of cancer cells, immune cells, and many other cell types contributing to a myriad of clinical outcomes for breast cancer (BrCa) patients. Chemokine receptors play an important role in maintaining the homeostasis of pro-inflammatory chemokines, and, in turn, help direct the migration of tumor-associated immune cells. Atypical chemokine receptors, such as the Atypical Chemokine Receptor 1 (ACKR1/DARC), act to sequester chemokine activity and control leukocyte migration during tumor-associated inflammation. ACKR1 is unique in that its associated gene carries a fixed mutation in African populations, causing the receptor to not be expressed on red blood cells as a response to endemic malaria in Africa. It is well-known that African-Americans in the United States tend to develop more aggressive BrCa subtypes, and as a result, experience more deaths per year from BrCa related causes. The purpose of this study is to characterize this disparity by investigating the distinct migration of tumor-associated immune cells between ACKR1 positive and negative tumors. We also wish to determine any correlations between ACKR1 and pro-inflammatory chemokines in various BrCa cell types. Using immunohistochemistry, relative expression levels were determined for ACKR1, cytotoxic T-cells, B-cells, dendritic cells, and macrophages in primary breast tumor samples. The levels of pro-inflammatory chemokines in circulation were determined using a Luminex immuno-assay on peripheral blood samples. Localization of ACKR1-associated chemokines was also investigated using immunofluorescence. Initial results from our study cohort indicate differential expression of immune cells on tumors expressing ACKR1, leading to a unique tumor microenvironment. We observed that ACKR1 positive tumors recruited B-cells and dendritic cells, whereas, ACKR1 negative tumors did not. Also, we found a positive correlation between ACKR1 expression in tumor tissue, and expression of CCL2 (MCP-1) and CXCL8 (IL-8) in the peripheral blood of our cancer patients. Finally, a strong co-localization of ACKR1 with CCL2 and CXCL8 was observed in cultured mammalian breast cancer cells. Overall, our pilot data suggests that the presence of ACKR1 on tumor cells changes the tumor microenvironment by recruiting a distinct subset of immune cells and pro-inflammatory chemokines to the area of inflammation. Citation Format: Brittany D. Jenkins, Rachel N. Martini, Rupali Hire, Michele A. Monteil, Melissa B. Davis. Distinct recruitment of tumor-associated immune cells correlates with increased pro-malignant chemokines in tumors expressing epithelial Atypical Chemokine Receptor 1 (ACKR1/DARC). [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B39.

Peptidylarginine deiminases (PADs) post-translationally convert arginine into neutral citrulline residues. Our past work shows that PADs are expressed in the canine and murine mammary glands; however, the mechanisms regulating PAD expression and the function of citrullination in the normal mammary gland are unclear. Therefore, the first objective herein was to investigate regulation of PAD expression in mammary epithelial cells. We first examined PAD levels in CID-9 cells, which were derived from the mammary gland of mid-pregnant mice. PAD3 expression is significantly higher than all other PAD isoforms and mediates protein citrullination in CID-9 cells. We next hypothesized that prolactin regulates PAD3 expression. To test this, CID-9 cells were stimulated with 5 μg/mL of prolactin for 48 hours which significantly increases PAD3 mRNA and protein expression. Use of a JAK2 inhibitor and a dominant negative (DN)-STAT5 adenovirus indicate that prolactin stimulation of PAD3 expression is mediated by the JAK2/STAT5 signaling pathway in CID-9 cells. In addition, the human PAD3 gene promoter is prolactin responsive in CID-9 cells. Our second objective was to investigate the expression and activity of PAD3 in the lactating mouse mammary gland. PAD3 expression in the mammary gland is highest on lactation day 9 and coincident with citrullinated proteins such as histones. Use of the PAD3 specific inhibitor, Cl4-amidine, indicates that PAD3, in part, can citrullinate proteins in L9 mammary glands. Collectively, our results show that upregulation of PAD3 is mediated by prolactin induction of the JAK2/STAT5 signaling pathway, and that PAD3 appears to citrullinate proteins during lactation.

Glucocorticoid hormones (CORT) are predicted to promote adaptation to variable environments, yet little is known about the potential for CORT secretion patterns to respond to selection in free-living populations. We assessed the heritable variation underlying differences in hormonal phenotypes using a cross-foster experimental design with nestling North American barn swallows ( Hirundo rustica erythrogaster ). Using a bivariate animal model, we partitioned variance in baseline and stress-induced CORT concentrations into their additive genetic and rearing environment components and estimated their genetic correlation. Both baseline and stress-induced CORT were heritable with heritability of 0.152 and 0.343, respectively. We found that the variation in baseline CORT was best explained by rearing environment, whereas the variation in stress-induced CORT was contributed to by a combination of genetic and environmental factors. Further, we did not detect a genetic correlation between these two hormonal traits. Although rearing environment appears to play an important role in the secretion of both types of CORT, our results suggest that stress-induced CORT levels are underlain by greater additive genetic variance compared with baseline CORT levels. Accordingly, we infer that the glucocorticoid response to stress has a greater potential for evolutionary change in response to selection compared with baseline glucocorticoid secretion patterns.