BackgroundHeterogeneity of immune gene expression patterns of luminal breast cancer (BC), which is clinically heterogeneous and overall considered as low immunogenic, has not been well studied especially in non-European populations. Here, we aimed at characterizing the immune gene expression profile of luminal BC in an Asian population and associating it with patient characteristics and tumor genomic features.MethodsWe performed immune gene expression profiling of tumor and adjacent normal tissue in 92 luminal BC patients from Hong Kong using RNA-sequencing data and used unsupervised consensus clustering to stratify tumors. We then used luminal patients from The Cancer Genome Atlas (TCGA, N = 564) and a Korean breast cancer study (KBC, N = 112) as replication datasets.ResultsBased on the expression of 130 immune-related genes, luminal tumors were stratified into three distinct immune subtypes. Tumors in one subtype showed higher level of tumor-infiltrating lymphocytes (TILs), characterized by T cell gene activation, higher expression of immune checkpoint genes, higher nonsynonymous mutation burden, and higher APOBEC-signature mutations, compared with other luminal tumors. The high-TIL subtype was also associated with lower ESR1/ESR2 expression ratio and increasing body mass index. The comparison of the immune profile in tumor and matched normal tissue suggested a tumor-derived activation of specific immune responses, which was only seen in high-TIL patients. Tumors in a second subtype were characterized by increased expression of interferon-stimulated genes and enrichment for TP53 somatic mutations. The presence of three immune subtypes within luminal BC was replicated in TCGA and KBC, although the pattern was more similar in Asian populations. The germline APOBEC3B deletion polymorphism, which is prevalent in East Asian populations and was previously linked to immune activation, was not associated with immune subtypes in our study. This result does not support the hypothesis that the germline APOBEC3B deletion polymorphism is the driving force for immune activation in breast tumors in Asian populations.ConclusionOur findings suggest that immune gene expression and associated genomic features could be useful to further stratify luminal BC beyond the current luminal A/B classification and a subset of luminal BC patients may benefit from checkpoint immunotherapy, at least in Asian populations.

A primary culture of mouse endometrial epithelium grown on permeable supports was established and the electrogenic ion transport across the endometrial epithelium was studied using the short-circuit current (I(SC)) technique. Enzymatically isolated mouse endometrial cells were immunostained with epithelial cells markers, cytokeratins, indicating an epithelial origin of the culture. Mouse endometrial epithelial cells grown on Millipore filters formed polarized monolayers with junctional complexes as revealed by light and electron microscopy. The cultured monolayers exhibited an average basal I(SC) of 4.6 +/- 0.3 microA/cm2, transepithelial voltage of 2.7 +/- 0.2 mV and transepithelial resistance of 599 +/- 30 omega cm2. The basal current was reduced by 85% in Na+-free solution and 13% in Cl(-)-free solution. The basal current could also be substantially (57.7%) blocked by an apical Na+ channel blocker, amiloride (10 microM), suggesting that Na+ absorption largely contributed to the basal current. Apical addition of Cl- channel blocker, DPC (2 mM), also exhibited an inhibitory effect, 19.4%, on the basal I(SC), indicating minor involvement of Cl- secretion as compared to that of Na+ absorption. The cultured endometrial epithelium also responded to a number of secretagogues including adrenaline and forskolin with increases in the I(SC), which could involve substantial Cl- secretion. The present study has established a culture of mouse endometrial epithelium exhibiting predominantly Na+ absorption under unstimulated condition, and Cl- secretion in response to various secretagogues. This culture may be useful for studying various regulatory mechanisms of electrogenic ion transport across the endometrial epithelium.

The beta-adrenergic (cAMP-dependent) regulation of Cl- conductance is defective in cystic fibrosis (CF). The present study explored alternative regulation of anion secretion in CF pancreatic ductal cells (CFPAC-1) by angiotensin II (AII) using the short-circuit current (ISC) technique. An increase in ISC could be induced in CFPAC-1 cells by basolateral or apical application of AII in a concentration-dependent manner (EC50 at 3 microm and 100 nm, respectively). Angiotensin receptor subtypes were identified using specific antagonists, losartan and PD123177, for AT1 and AT2 receptors, respectively. It was found that losartan (1 microm) could completely inhibit the AII-induced ISC, whereas, PD123177 exerted insignificant effect on the ISC, indicating predominant involvement of AT1 receptors. The presence of AT1 receptors in CFPAC-1 cells was also demonstrated by immunohistochemical studies using specific antibodies against AT1 receptors. Confocal microscopic study demonstrated a rise in intracellular Ca2+ upon stimulation by AII indicating a role of intracellular Ca2+ in mediating the AII response. Depletion of intracellular but not extracellular pool of Ca2+ diminished the AII-induced ISC. Treatment of the monolayers with a Cl- channel blocker, DIDS, markedly reduced the ISC, indicating that a large portion of the AII-activated ISC was Cl--dependent. AII-induced ISC was also observed in monolayers whose basolateral membranes had been permeabilized by nystatin, suggesting that the ISC was mediated by apical Cl- channels. Our study indicates an AT1-mediated Ca2+-dependent regulatory mechanism for anion secretion in CF pancreatic duct cells which may be important for the physiology and pathophysiology of the pancreas.

Disease heterogeneity of immune gene expression patterns of luminal breast cancer (BC) has not been well studied. We performed immune gene expression profiling of tumor and adjacent normal tissue in 92 Asian luminal BC patients and identified three distinct immune subtypes. Tumors in one subtype exhibited signs of T-cell activation, lower ESR1/ESR2 expression ratio and higher expression of immune checkpoint genes, nonsynonymous mutation burden, APOBEC-signature mutations, and increasing body mass index compared to other luminal tumors. Tumors in a second subtype were characterized by increased expression of interferonstimulated genes and enrichment for TP53 somatic mutations. The presence of three immune subtypes within luminal BC was replicated in cases drawn from The Cancer Genome Atlas and a Korean breast cancer study. Our findings suggest that immune gene expression and associated genomic features could be useful to further stratify luminal BC beyond the current luminal A/B classification.Introduction:

Different signal transduction pathways, i.e. Ca2+- and cAMP-dependent, involved in mediating the effects of angiotensin II (AII) were investigated separately using the short-circuit current (Isc) technique and radioimmunoassay (RIA) in a cystic fibrosis pancreatic cell line (CFPAC-1) which exhibits defective cAMP-dependent but intact Ca2+-dependent anion secretion. The AII-induced Isc could be inhibited by the specific antagonist for AT1, losartan (1 microM), but not the antagonist for AT2, PD123177 (up to 10 microM). The AII-induced Isc was also reduced by the treatment of the cells with a Ca2+ chelator, BAPTA-AM (100 microM), indicating a dependence of the AII-induced anion secretion on the intracellular Ca2+. Treatment of the cells with pertussis toxin (0.1 microg/ml) or a phospholipase C (PLC) inhibitor, U73122 (5 microM), resulted in a substantial reduction in the AII-induced Isc indicating involvement of Gi and PLC in the Ca2+-dependent anion secretion. RIA measurements showed that AII stimulated an increase in cAMP production which could be reduced by losartan, pertussis toxin and U73122 but not BAPTA-AM. In addition, inhibitors of cyclooxygenase, indomethacin (10 microM) and piroxicam (10 microM), did not have any effect on the AII-induced cAMP production, excluding the involvement of prostaglandins. Our results suggest that both AII-stimulated cAMP and Ca2+-dependent responses are mediated by the AT1 receptor and Gi-coupled PLC pathway. However, the AII-stimulated cAMP production in CFPAC-1 cells is not dependent on Ca2+ or the formation of prostaglandins.