Myoepithelial Cell Differentiation Research Articles

Clinical and Histopathologic Study of Apocrine-Type Mixed Tumor of the Skin.

Mixed tumor of the skin (MTS) is a tumor characterized by folliculosebaceous-apocrine differentiation. Because of the wide range of histological variations, understanding the unique features of MTS can help improve diagnosis. This study describes the histopathological characteristics of MTS, mainly apocrine-type MTS (AMT), using 166 cases of AMT. We found that nodular aggregates of myoepithelial cells, mucinous changes in the stroma, and follicular differentiation were standard characteristic features of MTS. Among the cases studied, 67% showed prominent follicular germinative cells and 40% showed prominent lipomatous metaplasia in the stroma. These cases often pose difficulties for the diagnosis of AMT because of insufficient evidence of sweat glands or myoepithelial cell differentiation. This is the first study to examine how the histological features of AMT change as the tumor extends deeper into the dermis. We found that the proportion of AMT with folliculosebaceous differentiation and large lumina increased as it got deeper into the dermis. Histopathological diagnosis of MTS is vital because the clinical symptoms lack specificity. This study enhances our understanding of the histopathological characteristics of MTS.

Isogenic Mammary Models of Intraductal Carcinoma Reveal Progression to Invasiveness in the Absence of a Non-Obligatory In Situ Stage.

In breast cancer, progression to invasive ductal carcinoma (IDC) involves interactions between immune, myoepithelial, and tumor cells. Development of IDC can proceed through ductal carcinoma in situ (DCIS), a non-obligate, non-invasive stage, or IDC can develop without evidence of DCIS and these cases associate with poorer prognosis. Tractable, immune-competent mouse models are needed to help delineate distinct mechanisms of local tumor cell invasion and prognostic implications. To address these gaps, we delivered murine mammary carcinoma cell lines directly into the main mammary lactiferous duct of immune-competent mice. Using two strains of immune-competent mice (BALB/c, C57BL/6), one immune-compromised (severe combined immunodeficiency; SCID) C57BL/6 strain, and six different murine mammary cancer cell lines (D2.OR, D2A1, 4T1, EMT6, EO771, Py230), we found early loss of ductal myoepithelial cell differentiation markers p63, α-smooth muscle actin, and calponin, and rapid formation of IDC in the absence of DCIS. Rapid IDC formation also occurred in the absence of adaptive immunity. Combined, these studies demonstrate that loss of myoepithelial barrier function does not require an intact immune system, and suggest that these isogenic murine models may prove a useful tool to study IDC in the absence of a non-obligatory DCIS stage-an under-investigated subset of poor prognostic human breast cancer.

ΔNp63 maintains the fidelity of the myoepithelial cell lineage and directs cell differentiation programs in the murine salivary gland

Salivary glands consist of several epithelial cell types of distinct lineages and functional characteristics that are established by directed differentiation programs of resident stem and progenitor cells. We have shown that ΔNp63, a crucial transcriptional regulator of stem/progenitor cells, is enriched in both the basal and myoepithelial cell (MEC) populations and that ΔNp63 positive cells maintain all the descendent epithelial cell lineages of the adult mouse salivary glands (mSGs). Although this pivotal role of ΔNp63 in driving the broader epithelial cell fate and identity in the mSG has been demonstrated, how ΔNp63 functions specifically in the commitment and differentiation of the MEC population is less understood. Using multiple genetic mouse models that allow for cell tracing, we show that ΔNp63 is critical in maintaining and renewing MECs, in part through the transcriptional regulation of Acta2 gene expression, a defining marker of this cell population. We demonstrate that during adult mSG homeostasis, ΔNp63 enriched MECs function as bipotent progenitor cells that maintain not only the MEC population, but also the distinctly different ductal cell lineages. The fidelity of this process is dependent on ΔNp63 expression, since MEC-specific ablation of ΔNp63 results in altered MEC differentiation and affects cellular plasticity resulting in aberrant differentiation of the intercalated ducts and acinar cells. In contrast, we find that the contribution of MECs to ductal and acinar cell regeneration following severe injury is independent of ΔNp63. Our observations offer new insights into cellular mechanisms driving MEC fate choices and differentiation programs in the context of salivary gland homeostasis and in response to injury and regeneration. Long term, these findings have implications for better treatment of salivary gland dysfunction through stem cell-based approaches.

Abstract IA034: Compromised myoepithelial cell differentiation correlates with DCIS to IDC transition

Abstract DCIS, breast cancer confined to mammary ducts, shares genomic and transcriptomic signatures with IDC. However for DCIS patients, standard of care for localized breast cancer remains controversial, as treatment reduces local recurrence but does not reduce risk of distant recurrence or mortality. Further, as the risk of dying from DCIS is ~5-20 fold less than from early stage breast cancer, questions remain concerning treatment harms. The fact that treatment does not reduce DCIS death rates provides indirect evidence that DCIS-stage tumor cells can disseminate and serve as reservoirs for metastatic disease. Since presence of myoepithelium is the clinical determinate of non-invasive disease, death from DCIS also implies that myoepithelial cell loss is not required for tumor cell dissemination. Here, we investigate whether changes in myoepithelial cell differentiation, rather than overt cell loss, associate with DCIS tumor cell dissemination. Using murine DCIS models and human DCIS tissue, our group reported sequential loss of myoepithelial cell differentiation markers p63, calponin, and SMA prior to progression to IDC. In human DCIS lesions delineated into low and high risk (pure DCIS vs mixed DCIS with IDC), loss of myoepithelial p63, calponin and SMA occurred more frequently in high risk DCIS lesions. Mice transgenic for calponin knockdown in the myoepithelium were at increased risk for mammary cancer. Combined, these data implicate loss in myoepithelial cell differentiation as a mediator of DCIS progression and dissemination. In human DCIS, compromised myoepithelium correlates with microinvasion and an immune infiltrate enriched in PD1+CD8 T cells. To address the role of immune cells in myoepithelial cell barrier function, we investigated 6 different immune competent mouse models of intraductal cancer (DCIS). While loss of myoepithelial differentiation markers were evident, IDC lesions developed in the absence of DCIS. Similarly, in immune compromised hosts, only IDC lesions were detected after intraductal injection of murine tumor cells, whereas human tumor cells formed stable DCIS lesions. In vitro, murine mammary tumor cells were more motile than human lines, and when tumor cells were co-cultured with myoepithelial cells, fewer myoepithelial to myoepithelial cell contacts were observed in the presence of the mouse tumor cells. Finally, in women, we find the differentiation state of mammary myoepithelium to be modulated by reproductive state, with myoepithelium during weaning–induced gland involution (an established risk window for breast cancer progression) sharing similarities with myoepithelium of high-risk DCIS lesions. In rodents, recently weaned hosts support increased DCIS to IDC transition. In sum, these studies implicate intrinsic and extrinsic regulators of myoepithelial cells, highlight the need for immune competent mouse models of DCIS, and raise the question of whether species mismatch in xenograft models impacts cellular crosstalk between myoepithelial and tumor cells critical to our understanding of the DCIS to IDC transition. Citation Format: Sarah M. Bernhardt, Elizabeth Mitchell, Tanya Russell, Sonali Jindal, Jayasri Narasimhan, Reuben J. Hoffmann, AeSoon Benson, Pepper Schedin. Compromised myoepithelial cell differentiation correlates with DCIS to IDC transition [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr IA034.

A parasympathetic neurotransmitter induces myoepithelial cell differentiation during salivary gland development

Myoepithelial cells (MECs) are responsible for receiving stimuli from the central nervous system and translating their responses into the form of secretion into glandular tissue, including salivary glands (SG), sweet glands, and mammary glands. SG MECs cause the secretion of serous saliva by contracting of acini/ductal cells with acetylcholine (Ach) from parasympathetic nerves via muscarinic receptors. To response the parasympathetic physiological stimulation, SG epithelial cell-derived MECs are supposed to be induced and placed adjacent to parasympathetic system nerve ends in SGs by forming a neuro-myoepithelial junction. For salivary secretion to function under parasympathetic control, therefore, specific regions of salivary gland epithelial cells must be mapped and the epithelium near the nerve must differentiate into MECs in order to form a nerve-myoepithelial junction during organogenesis. We hypothesized that the epithelium near the parasympathetic nerves is induced the differentiation into MECs by which the neurotransmitter acetylcholine via muscarinic receptors. qPCR and whole-mount immunohistochemical analysis in ex vivo organ culture system revealed that SG epithelial cells near a parasympathetic nerve were found to be induced to differentiate into MECs via the cholinergic receptor muscarinic 1 by carbachol (CCh), an acetylcholine agonist. In addition, CCh stimulated ERK and Akt signaling for the induction of MEC differentiation in rat submandibular gland epithelial cells. These findings indicate that muscarinic action is required for the induction of MECs and formation of a neuro-myoepithelial junction in developing SGs. This study proposes a novel concept for tissue architecture to form a neuro-myoepithelial junction during neurofunctional organogenesis including SGs.

Genetic heterogeneity and therapeutic target detection through microdissection in solid-type adenoid cystic carcinoma

Solid-type adenoid cystic carcinomas (ACCs) are highly aggressive and heterogeneous tumours. Because of their rarity, therapeutic strategies guided by genetic profiles based on next generation sequencing (NGS) have not been published for these tumours. Forty-nine solid-type ACCs including 43 tumours with a predominantly solid pattern, and six tumours comprising a roughly equal mixture of cribriform/tubular and solid histological forms were included in our study. The solid components from the 49 solid ACCs were enriched for mutations of genes in the NOTCH pathway (NOTCH1 61%, SPEN 24%) and chromatin remodelling pathway and the absence of myoepithelial cell differentiation. Cases with NOTCH1 mutations exhibited strong NICD expression, which was associated with poor overall and distant metastasis free survival. BRCA2 mutation and BCOR/BCORL1 mutations were observed in 20% and 18.4% of solid ACCs, respectively. In six of the solid ACCs, intratumour heterogeneity was delineated between the cribriform/tubular and solid components. NOTCH1 and FGFR2 mutations as well as NOTCH2 amplification were restricted to the solid component, indicating clonal selection within the same tumour. In two recurrent/metastatic solid ACCs, the subclones evolved in progression for local relapse and distant metastasis, although they manifested close genomic resemblance to primary tumours. Guided by the genetic profiles, the preclinical efficiency of the gamma-secretase inhibitor BMS-906024 was evaluated in patient derived xenograft models (PDXs) with activating NOTCH1 mutations and demonstrated robust antitumour effects. Our report revealed intratumour heterogeneity among solid-types within an ACC as well as the inter-tumour evolution of dominant clones among two primary and recurrent/metastatic tumours. In contrast to cribriform/tubular ACCs, solid-type ACCs should be approached with a distinct therapeutic strategy, particularly targeting NOTCH1. Microdissecting the highest grade component guided by histology is a highly recommended tumour sampling strategy and facilitates the detection of key molecular targets.

Proliferative Activity of Myoepithelial Cells in of Mucoepidermoid Carcinoma

AIM: The aim of the study is to investigate the role of myoepithelial cells in the pathogenesis of mucoepidermoid carcinoma (MEC) using the double immunohistochemical staining; α _smooth muscle actin (_α-SMA)as specific marker for the myoepithelial cell differentiation and proliferative cell nuclear antigen (PCNA) as a marker for proliferative activity of myoepithelial cells. MATERIAL AND METHODS: Retrospective study of twenty salivary gland specimens (ten MEC and ten normal salivary glands) were studied using double immunohistochemical labeling for α _smooth muscle actin α-SMA) and proliferative cell nuclear antigen (PCNA). The SPSS statistical package was used for data analysis (IBM SPSS Statistics for Windows, Version 20.0, Released 2011, IBM Corp, and Armonk, NY, USA). RESULTS: In mucoepidermoid carcinomas, no positivity of α-SMA was seen in neoplastic cells (Frequent test), and it was just observed in the stroma of tumor, in the walls of blood vessels whereas, PCNA was positive, especially in high-grade tumors. In contrast, in normal salivary glands, the proliferating myoepithelial cells are stained by both α-SMA and PCNA. CONCLUSIONS: We believe that the myoepithelial cells have no a role in the development of mucoepidermoid carcinoma.

Cell signaling regulation in salivary gland development.

The mammalian salivary gland develops as a highly branched structure designed to produce and secrete saliva. This review focuses on research conducted on mammalian salivary gland development, particularly on the differentiation of acinar, ductal, and myoepithelial cells. We discuss recent studies that provide conceptual advances in the understanding of the molecular mechanisms of salivary gland development. In addition, we describe the organogenesis of submandibular glands (SMGs), model systems used for the study of SMG development, and the key signaling pathways as well as cellular processes involved in salivary gland development. The findings from the recent studies elucidating the identity of stem/progenitor cells in the SMGs, and the process by which they are directed along a series of cell fate decisions to form functional glands, are also discussed. Advances in genetic tools and tissue engineering strategies will significantly increase our knowledge about the mechanisms by which signaling pathways and cells establish tissue architecture and function during salivary gland development, which may also be conserved in the growth and development of other organ systems. An increased knowledge of organ development mechanisms will have profound implications in the design of therapies for the regrowth or repair of injured tissues. In addition, understanding how the processes of cell survival, expansion, specification, movement, and communication with neighboring cells are regulated under physiological and pathological conditions is critical to the development of future treatments.

IGF1R constitutive activation expands luminal progenitors and influences lineage differentiation during breast tumorigenesis

Breast tumors display tremendous heterogeneity in part due to varying molecular alterations, divergent cells of origin, and differentiation. Understanding where and how this heterogeneity develops is likely important for effective breast cancer eradication. Insulin-like growth factor (IGF) signaling is critical for normal mammary gland development and function, and has an established role in tumor development and resistance to therapy. Here we demonstrate that constitutive activation of the IGF1 receptor (IGF1R) influences lineage differentiation during mammary tumorigenesis. Transgenic IGF1R constitutive activation promotes tumors with mixed histologies, multiple cell lineages and an expanded bi-progenitor population. In these tumors, IGF1R expands the luminal-progenitor population while influencing myoepithelial differentiation. Mammary gland transplantation with IGF1R-infected mammary epithelial cells (MECs) resulted in hyperplastic, highly differentiated outgrowths and attenuated reconstitution. Restricting IGF1R constitutive activation to luminal versus myoepithelial lineage-sorted MECs resulted in ductal reconstitutions co-expressing high IGF1R levels in the opposite lineage of origin. Using in vitro models, IGF1R constitutively activated MCF10A cells showed increased mammosphere formation and CD44+/CD24-population, which was dependent upon Snail and NFκB signaling. These results suggest that IGF1R expands luminal progenitor populations while also stimulating myoepithelial cell differentiation. This ability to influence lineage differentiation may promote heterogeneous mammary tumors, and have implications for clinical treatment.

Loss of myoepithelial calponin-1 characterizes high-risk ductal carcinoma in situ cases, which are further stratified by T cell composition.

A hallmark of ductal carcinoma in situ (DCIS) progression is a loss of the surrounding ductal myoepithelium. However, whether compromise in myoepithelial differentiation, rather than overt cellular loss, can be used to predict the risk of DCIS progression is unknown. Here we address this question utilizing pure and mixed DCIS cases (N = 30) as surrogates for DCIS at low and high risk for progression, respectively. We used multiplex immunohistochemical staining to evaluate the relationship between myoepithelial cell differentiation and lymphoid immune cell types associated with poor prognostic DCIS. Our results show that myoepithelial calponin‐1 discriminates between pure and mixed DCIS lesions better than histological subtype, presence of necrosis, or nuclear grade. Additionally, focal loss of myoepithelial cells associated with increased PD‐1+CD8+ T cells, which suggests a link between the myoepithelium and immune surveillance. To identify associations between calponin‐1 expression and immune response, we performed unsupervised hierarchical clustering of myoepithelial and immune cell biomarkers on 219 DCIS lesions from 30 cases. Notably, the majority of pure (low‐risk) DCIS lesions clustered in a high calponin‐1, T cell low group, whereas the majority of mixed (high‐risk) DCIS lesions clustered in a low calponin‐1, T cell high group, specifically with CD8+ and PD‐1+CD8+ T cells. However, a subset of pure DCIS lesions had a similar calponin‐1 and immune signature as the majority of mixed DCIS lesions, which have low calponin‐1 and T cell enrichment—raising the possibility that these pure DCIS lesions might be at a high risk for progression.

Perturbed myoepithelial cell differentiation in BRCA mutation carriers and in ductal carcinoma in situ

Myoepithelial cells play key roles in normal mammary gland development and in limiting pre-invasive to invasive breast tumor progression, yet their differentiation and perturbation in ductal carcinoma in situ (DCIS) are poorly understood. Here, we investigated myoepithelial cells in normal breast tissues of BRCA1 and BRCA2 germline mutation carriers and in non-carrier controls, and in sporadic DCIS. We found that in the normal breast of non-carriers, myoepithelial cells frequently co-express the p63 and TCF7 transcription factors and that p63 and TCF7 show overlapping chromatin peaks associated with differentiated myoepithelium-specific genes. In contrast, in normal breast tissues of BRCA1 mutation carriers the frequency of p63+TCF7+ myoepithelial cells is significantly decreased and p63 and TCF7 chromatin peaks do not overlap. These myoepithelial perturbations in normal breast tissues of BRCA1 germline mutation carriers may play a role in their higher risk of breast cancer. The fraction of p63+TCF7+ myoepithelial cells is also significantly decreased in DCIS, which may be associated with invasive progression.

ACTIVATING NOTCH1 MUTATION IN HIGH-GRADE EVOLUTION OF ADENOID CYSTIC CARCINOMA

Objective Salivary adenoid cystic carcinoma(SACC) is identified as a tumor with biphasic differentiation of epithelial and myoepithelial cells, showing tubular, cribriform and solid subtypes. The solid subtype is considered as high-grade with more aggressiveness and poorer prognosis. However, the molecular mechanism remains unknown. The aim of this study was to identify the clinicopathological characteristics of high-grade SACC, to clarify the molecular mechanism underline its distinct characteristics, and hopefully to explore potential molecular targets for SACC therapy. Study design Activated Notch1 (NICD) and myoepithelial cell markers were used for immunohistochemistry in 119 SACCs including 59 cribriform-tubular and 60 solid subtypes. Notch1 mutations were analyzed by DNA sequencing in all the SACC cases. The effect of activating NOTCH pathway on the biological behavior of SACC cell lines was investigated with transfection and functional studies. Results Notch1 mutations in the negative regulatory region and Pro-Glu-Ser-Thr–rich domains were identified in 26 of 119 patients with SACC, and 24 (92%) of 26 Notch1 mutant cases were predicted to be activating with NICD positive, with 2 cases predicted to be inactivating with NICD negative. Most (23/24, 96%) cases with activating Notch1 mutations were high-grade solid SACCs. Meantime, only 17 (18%) of 93 NOTCH1 wild-type tumors stained positive, and 16 of 17 tumors with NICD positive were high-grade solid subtypes. Furthermore, high-grade solid SACCs showed dramatically decreased short-term survival, tended to suffer bone invasion and metastasis, and presented NICD positive and myoepithelial cell markers negative simultaneously. Transfection and functional studies showed forced NICD expression promoted high level of proliferation and migration in SACC cells. Conclusions Our findings showed activating Notch1 mutations were related to the loss of myoepithelial differentiation in high-grade SACCs and might contribute to higher proliferation and worse outcome in high-grade tumors. Targeting the Notch signaling pathway in high-grade SACCs may provide therapeutic benefits.

Abstract B62: Perturbed myoepithelial cell differentiation in BRCA mutation carriers and in DCIS (ductal carcinoma in situ)

Abstract The normal mammary gland is composed of multiple cell types, including luminal and myoepithelial cells. Myoepithelial cells have also been referred to as natural tumor suppressors due to their inhibitory effect on various neoplastic phenotypes, including tumor cell growth, invasion, and angiogenesis. We have previously determined that DCIS-associated myoepithelial cells lack clonal genetic aberrations, but their DNA methylation and gene expression patterns are distinct from that of normal myoepithelial cells, implying perturbed differentiation. We also showed that myoepithelial cells play key roles in preventing tumor growth and invasive progression in experimental models of DCIS, and in immune escape in breast cancer. These data support the hypothesis that the progressive loss of normal myoepithelial cell function in DCIS is a key step in the transition to invasive breast carcinomas, and assessing myoepithelial markers may identify patients with high risk of progression. However, the mechanisms underlying myoepithelial cell aberrations and eventual loss in DCIS are not well understood. In the present study, we used a combination of genomic profiling of human tissue samples and functional assays in a DCIS model to investigate regulators of normal myoepithelial cells and perturbations of these in BRCA mutation carriers and in DCIS. We first explored cellular heterogeneity of the CD10+ cell population in normal human breast tissues of nulliparous and parous women, from reduction mammoplasties and BRCA1 and BRCA2 mutation carriers undergoing prophylactic mastectomy. We identified two distinct CD10+ cell populations distinguished by the expression of CD44 and characterized their gene expression profiles. We also profiled CD10+ cells from BRCA1 and BRCA2 mutation carriers compared to normal noncarriers and found a clear separation of three distinct groups reflecting germline mutation status. Next we defined the p63 cistrome and the enhancer landscape of normal myoepithelial cells and identified alterations of these due to germline BRCA mutation status. Based on our initial results, we focused in p63 and TCF7 as these two transcription factors appear to be central for establishing myoepithelial cell fate. To address the functional relevance of p63 and TCF7 in myoepithelial cell differentiation, we used the MCFDCIS xenograft model of DCIS. These results support the hypothesis that myoepithelial cells play key roles in invasive progression and have significant impact on our understanding of DCIS progression and the increased breast cancer risk of BRCA mutation carriers. Citation Format: Lina Ding, Ying Su, Muhammad Ekram, Sung Jin Huh, Noga Bloushtain-Qimron, Sibgat Choudhury, William Hines, Jun Yao, Mina Bissell, Kornelia Polyak. Perturbed myoepithelial cell differentiation in BRCA mutation carriers and in DCIS (ductal carcinoma in situ) [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B62.

CDKN2A loss and PIK3CA mutation in myoepithelial-like metaplastic breast cancer.

Metaplastic breast carcinoma comprises a heterogeneous group of tumours with poorly understood pathogenesis. A subset of metaplastic breast cancers show myoepithelial differentiation and constitute a morphological spectrum with ill-defined borders from fibromatosis-like spindle cell carcinoma to myoepithelial carcinoma. In a series of 34 metaplastic breast cancers with spindle cell and myoepithelial differentiation, we found recurrent genetic aberrations, which set them apart from other metaplastic breast cancers and suggest a unique pathogenesis. The majority of cases (28 of 34 patients; 82.4%) showed distinct chromosomal loss in the 9p21.3 region, including CDKN2A and CDKN2B. Biallelic loss of the CDKN2A/B region was found in 50% of deleted cases. Expression of the cyclin-dependent kinase inhibitor CDKN2A (p16) was missing in all samples affected by 9p21.3 loss. Other genomic alterations frequently occurring in triple-negative and metaplastic breast cancer were absent or found in only a minority of cases. Gains of whole chromosome 5 and chromosomal region 5p were observed in nine cases, and were associated with recurrences (p < 0.001). In 64.3% of cases, 9p21.3 loss was accompanied by concurrent PIK3CA mutation. Both genomic abnormalities were also detectable in adenomyoepitheliomas (4/12), which are considered to represent the precursor lesion of myoepithelial metaplastic breast cancer. In adenomyoepithelioma, PIK3CA mutation was present in both luminal epithelial and myoepithelial cells, whereas p16 loss was found only in the latter. We conclude that 9p21.3 (CDKN2A) loss and PIK3CA mutation characterize a subgroup of metaplastic breast cancers with myoepithelial and spindle cell differentiation. Myoepithelial cells in adenomyoepithelioma may show identical aberrations. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A METHOD FOR QUANTIFICATION OF CALPONIN EXPRESSION IN MYOEPITHELIAL CELLS IN IMMUNOHISTOCHEMICAL IMAGES OF DUCTAL CARCINOMA IN SITU.

Ductal carcinoma in situ (DCIS) is breast cancer confined within mammary ducts, surrounded by an intact myoepithelial cell layer that prevents local invasion. A DCIS diagnosis confers increased lifetime risk of developing invasive breast cancer (IBC) and results in surgical excision with radiation, and possibly endocrine- or chemo-therapy. DCIS is known to be over treated, with associated co-morbidities. Biomarkers are needed that delineate patients at low risk of DCIS progression from patients requiring more aggressive treatment. Investigating the role of myoepithelial cell differentiation in barrier function is anticipated to provide insight into DCIS progression and delineate between low and high risk lesions. Here, we develop a high throughput technique to assess loss of myoepithelial differentiation markers. This method facilitates automated analysis of a clinically relevant histopathologic feature, as demonstrated by a high correlation with pathologist annotation (r = 0.959), and further, contributes analytical foundations to a multiplexed immunohistochemistry (IHC) approach.

Multiplex Immunohistochemistry Provides Insight Into Cross‐Talk Between Myoepithelial And Immune Cells In Ductal Carcinoma In Situ (DCIS) Progression

Every year in the U.S. ~50,000 women are diagnosed with Ductal Carcinoma in Situ (DCIS). Long term studies of untreated DCIS show ~30% of women progressed to invasive breast cancer (IBC), providing strong rational for treating DCIS similar to early stage IBC. Disease management entails breast‐conserving surgery (BCS) in ~70% of women with or without adjuvant radiation therapy (RT), or unilateral and/or bilateral mastectomy in about 30% of women. While RT and mastectomy reduce risk of IBC or DCIS ipsilateral recurrence, support of potential overtreatment is suggested by long term outcome studies demonstrating low mortality from DCIS (1–2% at 10 years) regardless of treatment received. Thus, prognostic markers that can distinguish DCIS at high versus low risk of progression are needed to personalize treatment decisions. The tumor microenvironment, including myoepithelial and immune cells, has been shown by us and others to play a pivotal role in tumor progression, and we have advanced murine models to study human DCIS progression. In an immune compromised model, the loss of myoepithelial cell calponin, a regulatory component of smooth muscle contraction, specifically corresponded with a ~130‐fold increased probability of adjacent human tumor cells expressing the poor prognostic tumor biomarker p63; implicating calponin as a tumor suppressor protein that acts on adjacent tumor cells. Using immune competent murine models, we find faster progression to invasive tumors, suggesting a supportive role for the immune system in DCIS to IBC progression. We hypothesized that pro‐tumorigenic immune cells will be found near DCIS lesions that have lost myoepithelial differentiation markers and/or tumor cells that have a poor‐prognostic basal phenotype. To test this hypothesis, we have developed novel computer‐assisted computational methods to characterize the integrity of the myoepithelial cell layer in DCIS lesions and to provide near‐neighbor analysis to identify proximity to immune cells. This method involves image segmentation to identify the myoepithelial cell layer using the combined IHC images of SMA, calponin, and CK18, followed by geometric quantification to determine relative expression as well as physical proximity between distinct biomarker (Figure 1). We validated the ability of this computational method to determine loss of myoepithelial cell‐differentiation markers by comparing results to a pathologist's visual annotation, and found a Pearson's‐correlation coefficient of R = 0.959. Utilizing FFPE breast tissues from a cohort of women diagnosed with DCIS (OHSU IRB #15514), we performed multiplex sequential immunohistochemical (IHC) staining for markers of myoepithelial differentiation state (p63, SMA, calponin), immune cells (lymphoid: CD45, CD8, CD3, Foxp3, Granzyme B, PD‐1) and prognostic biomarkers (cyclooxygenase‐2, p16, ki67, ER, PR, Her‐2, tenascin‐c, cytokeratin 5). Our results show that loss of the myoepithelial differentiation marker calponin is associated with a relative decrease in CD4+Foxp3+ T regulatory cells and a concurrent increase in CD8+PD‐1+ T cells. These data suggest that 1) during DCIS progression there may be activation of cytotoxic effectors and concomitant decrease in Tregs to support tumor suppression, and 2) myoepithelial cell integrity may facilitate the activation state of local immune cells. Future studies include assessment for the role of myeloid lineages in myoepithelial cell integrity and DCIS progression.Support or Funding InformationThis research work is supported by OHSU Knight Cancer Institute – Borchard Early Investigator Award to SJ, OHSU OCTRI ‐ NW Kaiser specimen retrieval opportunity to PS and SJ, Susan G. Komen Post‐Doctoral fellowship to EM (PDF17480342).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Par-1b is required for morphogenesis and differentiation of myoepithelial cells during salivary gland development

ABSTRACTThe salivary epithelium initiates as a solid mass of epithelial cells that are organized into a primary bud that undergoes morphogenesis and differentiation to yield bilayered acini consisting of interior secretory acinar cells that are surrounded by contractile myoepithelial cells in mature salivary glands. How the primary bud transitions into acini has not been previously documented. We document here that the outer epithelial cells subsequently undergo a vertical compression as they express smooth muscle α-actin and differentiate into myoepithelial cells. The outermost layer of polarized epithelial cells assemble and organize the basal deposition of basement membrane, which requires basal positioning of the polarity protein, Par-1b. Whether Par-1b is required for the vertical compression and differentiation of the myoepithelial cells is unknown. Following manipulation of Par-1b in salivary gland organ explants, Par-1b-inhibited explants showed both a reduced vertical compression of differentiating myoepithelial cells and reduced levels of smooth muscle α-actin. Rac1 knockdown and inhibition of Rac GTPase function also inhibited branching morphogenesis. Since Rac regulates cellular morphology, we investigated a contribution for Rac in myoepithelial cell differentiation. Inhibition of Rac GTPase activity showed a similar reduction in vertical compression and smooth muscle α-actin levels while decreasing the levels of Par-1b protein and altering its basal localization in the outer cells. Inhibition of ROCK, which is required for basal positioning of Par-1b, resulted in mislocalization of Par-1b and loss of vertical cellular compression, but did not significantly alter levels of smooth muscle α-actin in these cells. Overexpression of Par-1b in the presence of Rac inhibition restored basement membrane protein levels and localization. Our results indicate that the basal localization of Par-1b in the outer epithelial cells is required for myoepithelial cell compression, and Par-1b is required for myoepithelial differentiation, regardless of its localization.

Pleomorphic adenoma and adenoid cystic carcinoma of salivary glands: E-cadherin immunoexpression and analysis of the CDH1 -160C/A polymorphism

ObjectiveDespite their similar cellular origin, pleomorphic adenomas (PA) and adenoid cystic carcinomas (ACC) present distinct behaviors. This study aimed to analyze the immunoexpression of E-cadherin in PA and ACC of salivary glands, and to investigate differences in its expression in relation to E-cadherin gene (CDH1) -160C/A polymorphism. DesignTwenty-four PA (15 cell-rich and 9 cell-poor tumors) and 24 ACC (10 tubular, 8 cribriform and 6 solid tumors) were selected for the analysis of pattern of distribution, and cellular localization of E-cadherin. In addition, E-cadherin expression was evaluated using the H-score scoring system. The CDH1 -160C/A polymorphism was investigated by PCR-RFLP. ResultsNo significant differences in pattern of distribution (p=0.181) and cellular localization (p=0.192) of E-cadherin were observed between PA and ACC. Comparison of PA and ACC cases revealed a higher median H-score in the latter (p=0.036). Cell-rich PA presented a higher H-score than cell-poor tumors (p=0.013), whereas no significant differences in E-cadherin expression were observed between ACC subtypes (p=0.254). The heterozygous genotype of the CDH1 -160C/A polymorphism was detected in only one PA and one ACC. H-scores for tumors carrying the polymorphism were below the lower quartile of their respective groups. ConclusionsThe results suggest that E-cadherin expression in PA and ACC is mainly related to cellular composition (epithelial cells versus myoepithelial cells) and degree of differentiation of myoepithelial cells in these tumors. The CDH1 -160C/A polymorphism does not seem to significantly influence the expression of E-cadherin in PA and ACC of salivary glands.

Myoepithelial Cell Differentiation Markers in Ductal Carcinoma in Situ Progression

We describe a preclinical model that investigates progression of early-stage ductal carcinoma in situ (DCIS) and report that compromised myoepithelial cell differentiation occurs before transition to invasive disease. Human breast cancer MCF10DCIS.com cells were delivered into the mouse mammary teat by intraductal injection in the absence of surgical manipulations and accompanying wound-healing confounders. DCIS-like lesions developed throughout the mammary ducts with full representation of human DCIS histologic patterns. Tumor cells were incorporated into the normal mammary epithelium, developed ductal intraepithelial neoplasia and DCIS, and progressed to invasive carcinoma, suggesting the model provides a rigorous approach to study early stages of breast cancer progression. Mammary glands were evaluated for myoepithelium integrity with immunohistochemical assays. Progressive loss of the myoepithelial cell differentiation markers p63, calponin, and α-smooth muscle actin was observed in the mouse myoepithelium surrounding DCIS-involved ducts. p63 loss was an early indicator, calponin loss intermediate, and α-smooth muscle actin a later indicator of compromised myoepithelium. Loss of myoepithelial calponin was specifically associated with gain of the basal marker p63 in adjacent tumor cells. In single time point biopsies obtained from 16 women diagnosed with pure DCIS, a similar loss in myoepithelial cell markers was observed. These results suggest that further research is warranted into the role of myoepithelial cell p63 and calponin expression on DCIS progression to invasive disease.

Basal cytokeratin phenotypes of myoepithelial cells indicates the origin of ductal carcinomas in situ of the breast.

Terminal duct lobular unit (TDLU) is widely accepted as the origin of ductal carcinoma in situ of breast. The differentiation states of myoepithelial cells of breast ductal system hint the development of breast hyperplastic lesions. Basal cytokeratin (CK) phenotypes indicate the differentiation of myoepithelial cells. Using antibodies of CK5/6, CK14, and CK17, this study reports the basal CK phenotypes of myoepithelial cells in 20 foci of normal breast, 20 usual ductal hyperplasias, 36 ductal carcinomas in situ (DCIS), and 28 sclerosing adenosis (SA). The results showed that the positive staining of basal CKs of myoepithelial cells in normal ducts were significantly higher than those in normal lobules. The basal CK expression of myoepithelial cells of DCIS and usual ductal hyperplasia was similar to that of normal duct, whereas that of SA was similar to that of normal lobule. We propose a modified model of TDLU origin of intraductal carcinoma that most of DCIS originate from terminal ducts of TDLU, whereas most SA originate from lobules.