Uterine leiomyosarcoma (uLMS) is a rare and aggressive gynaecological malignancy. Surgical removal and chemotherapy are commonly used to treat uLMS, but recurrence rates are high. Over the last few ...decades, clarification of the genomic landscape of uLMS has revealed a number of recurring mutations, including
,
,
and
. Such genomic aberrations are difficult to target therapeutically or are actively targeted in other malignancies, and their potential as targets for the treatment of uLMS remains largely unexplored. Recent identification of deficiencies in homologous recombination in a minority of these tumours, however, has provided a rationale for investigation of PARP inhibitors in this sub-set. Here, we review these mutations and the evidence for therapeutic avenues that may be applied in uLMS. We also provide a comprehensive background on diagnosis and current therapeutic strategies as well as reviewing preclinical models of uLMS, which may be employed not only in testing emerging therapies but also in understanding this challenging and deadly disease.
Estrogen induces proliferation of breast epithelial cells and is responsible for breast development at puberty. This tightly regulated control is lost in estrogen-receptor-positive (ER+) breast ...cancers, which comprise over 70% of all breast cancers. Currently, breast cancer diagnosis and treatment considers only the α isoform of ER; however, there is a second ER, ERβ. Whilst ERα mediates estrogen-driven proliferation of the normal breast in puberty and breast cancers, ERβ has been shown to exert an anti-proliferative effect on the normal breast. It is not known how the expression of each ER (alone or in combination) correlates with the ability of estrogen to induce proliferation in the breast. We assessed the levels of each ER in normal mouse mammary glands subdivided into proliferative and non-proliferative regions. ERα was most abundant in the proliferative regions of younger mice, with ERβ expressed most abundantly in old mice. We correlated this expression profile with function by showing that the ability of estrogen to induce proliferation was reduced in older mice. To show that the ER profile associated with breast cancer risk, we assessed ER expression in parous mice which are known to have a reduced risk of developing ERα breast cancer. ERα expression was significantly decreased yet co-localization analysis revealed ERβ expression increased with parity. Parous mice had less unopposed nuclear ERα expression and increased levels of ERβ. These changes suggest that the nuclear expression of ERs dictates the proliferative nature of the breast and may explain the decreased breast cancer risk with parity.
Background
Breast cancer (BCa) mortality is decreasing with early detection and improvement in therapies. The incidence of BCa, however, continues to increase, particularly estrogen-receptor-positive ...(ER +) subtypes. One of the greatest modifiers of ER + BCa risk is childbearing (parity), with BCa risk halved in young multiparous mothers. Despite convincing epidemiological data, the biology that underpins this protection remains unclear. Parity-induced protection has been postulated to be due to a decrease in mammary stem cells (MaSCs); however, reports to date have provided conflicting data.
Methods
We have completed rigorous functional testing of repopulating activity in parous mice using unfractionated and MaSC (CD24
mid
CD49f
hi
)-enriched populations. We also developed a novel serial transplant method to enable us to assess self-renewal of MaSC following pregnancy. Lastly, as each pregnancy confers additional BCa protection, we subjected mice to multiple rounds of pregnancy to assess whether additional pregnancies impact MaSC activity.
Results
Here, we report that while repopulating activity in the mammary gland is reduced by parity in the unfractionated gland, it is not due to a loss in the classically defined MaSC (CD24
+
CD49f
hi
) numbers or function. Self-renewal was unaffected by parity and additional rounds of pregnancy also did not lead to a decrease in MaSC activity.
Conclusions
Our data show instead that parity impacts on the stem-like activity of cells outside the MaSC population.
Ovarian fibromas and adenofibromas are rare ovarian tumours. They are benign tumours composed of spindle-like stromal cells (pure fibroma) or a mixture of fibroblast and epithelial components ...(adenofibroma). We have previously shown that 40% of benign serous ovarian tumours are likely primary fibromas due to the neoplastic alterations being restricted to the stromal compartment of these tumours. We further explore this finding by comparing benign serous tumours to pure fibromas.
Performing copy number aberration (CNA) analysis on the stromal component of 45 benign serous tumours and 8 pure fibromas, we have again shown that trisomy of chromosome 12 is the most common aberration in ovarian fibromas. CNAs were more frequent in the pure fibromas than the benign serous tumours (88% vs 33%), however pure fibromas more frequently harboured more than one CNA event compared with benign serous tumours. As these extra CNA events observed in the pure fibromas were unique to this subset our data indicates a unique tumour evolution. Gene expression analysis on the two cohorts was unable to show gene expression changes that differed based on tumour subtype. Exome analysis did not reveal any recurrently mutated genes.
Estrogen stimulates breast development during puberty and mammary tumors in adulthood through estrogen receptor-α (ERα). These effects are proposed to occur via ERα+ luminal cells and not the mammary ...stem cells (MaSCs) that are ERαneg. Since ERα+ luminal cells express stem cell antigen-1 (SCA-1), we sought to determine if SCA-1 could define an ERα+ subset of EpCAM+/CD24+/CD49fhi MaSCs. We show that the MaSC population has a distinct SCA-1+ population that is abundant in pre-pubertal mammary glands. The SCA-1+ MaSCs have less stem cell markers and less in vivo repopulating activity than their SCA-1neg counterparts. However, they express ERα and specifically enter the cell cycle at puberty. Using estrogen-deficient aromatase knockouts (ArKO), we showed that the SCA-1+ MaSC could be directly modulated by estrogen supplementation. Thus, SCA-1 enriches for an ERα+, estrogen-sensitive subpopulation within the CD24+/CD49fhi MaSC population that may be responsible for the hormonal sensitivity of the developing mammary gland.
•SCA-1+ delineates ER-positive cells in the CD24+ CD49fhi mammary stem population•SCA-1+ cells have lower repopulation activity•SCA-1+ cells are estrogen responsive
Mouse mammary stem cells are thought to be estrogen-receptor negative and receive hormonal influence via estrogen-receptor-positive luminal neighbors. In this article, Britt and colleagues describe a population within the mammary stem cell-enriched compartment that is estrogen-receptor positive and directly responsive to estrogens. This has implications for understanding how aberrant hormone exposure affects breast cancer risk.
Estrogen stimulates breast development during puberty and mammary tumors in adulthood through estrogen receptor-α (ERα). These effects are proposed to occur via ERα
luminal cells and not the mammary ...stem cells (MaSCs) that are ERα
. Since ERα
luminal cells express stem cell antigen-1 (SCA-1), we sought to determine if SCA-1 could define an ERα
subset of EpCAM
/CD24
/CD49f
MaSCs. We show that the MaSC population has a distinct SCA-1
population that is abundant in pre-pubertal mammary glands. The SCA-1
MaSCs have less stem cell markers and less in vivo repopulating activity than their SCA-1
counterparts. However, they express ERα and specifically enter the cell cycle at puberty. Using estrogen-deficient aromatase knockouts (ArKO), we showed that the SCA-1
MaSC could be directly modulated by estrogen supplementation. Thus, SCA-1 enriches for an ERα
, estrogen-sensitive subpopulation within the CD24
/CD49f
MaSC population that may be responsible for the hormonal sensitivity of the developing mammary gland.
Breast cancer (BCa) incidence increases following aberrant hormone exposure, which has been linked to direct effects on estrogen receptor (ER)
mammary epithelium. While estrogen exposure during ...mammary involution has been shown to drive tumour growth via neutrophils, the potential for the ER + immune microenvironment to mediate part (in addition to mammary epithelial cells) of hormonally controlled BCa risk during normal development has not been assessed. We collected mammary tissue, lymph nodes and blood from tumour naïve mice treated with, oophorectomy, estrogen (17β estradiol) or Fulvestrant. Flow cytometry was used to examine the impact on the frequency of innate and adaptive immune cells. Oophorectomy and fulvestrant decreased the proportion of macrophages, particularly pro-tumour polarized M2 macrophages and neutrophils. Conversely, dendritic cells were increased by these therapies, as were eosinophils. Estrogen increased the proportion of M2 macrophages and to a lesser extent CD4-CD8- double negative and FoxP3
regulatory T cells but decreased CD8 + T cells and B cells. Excluding eosinophils, these changes were restricted to the mammary tissue. This suggests that inhibiting estrogen action lowers the immune suppressive myeloid cells, increases in antigen presentation and eosinophil-mediated direct or indirect cytotoxic effects. In contrast, estrogen exposure, which drives BCa risk, increases the suppressive myeloid cells and reduces anti-tumour cytotoxic T cells. The impact of hormonal exposure on BCa risk, may in part be linked to its immune modulatory activity.
Ovarian carcinosarcoma (OCS) is an aggressive and rare tumor type with limited treatment options. OCS is hypothesized to develop via the combination theory, with a single progenitor resulting in ...carcinomatous and sarcomatous components, or alternatively via the conversion theory, with the sarcomatous component developing from the carcinomatous component through epithelial-to-mesenchymal transition (EMT). In this study, we analyzed DNA variants from isolated carcinoma and sarcoma components to show that OCS from 18 women is monoclonal. RNA sequencing indicated that the carcinoma components were more mesenchymal when compared with pure epithelial ovarian carcinomas, supporting the conversion theory and suggesting that EMT is important in the formation of these tumors. Preclinical OCS models were used to test the efficacy of microtubule-targeting drugs, including eribulin, which has previously been shown to reverse EMT characteristics in breast cancers and induce differentiation in sarcomas. Vinorelbine and eribulin more effectively inhibited OCS growth than standard-of-care platinum-based chemotherapy, and treatment with eribulin reduced mesenchymal characteristics and N-MYC expression in OCS patient-derived xenografts. Eribulin treatment resulted in an accumulation of intracellular cholesterol in OCS cells, which triggered a downregulation of the mevalonate pathway and prevented further cholesterol biosynthesis. Finally, eribulin increased expression of genes related to immune activation and increased the intratumoral accumulation of CD8+ T cells, supporting exploration of immunotherapy combinations in the clinic. Together, these data indicate that EMT plays a key role in OCS tumorigenesis and support the conversion theory for OCS histogenesis. Targeting EMT using eribulin could help improve OCS patient outcomes.
Genomic analyses and preclinical models of ovarian carcinosarcoma support the conversion theory for disease development and indicate that microtubule inhibitors could be used to suppress EMT and stimulate antitumor immunity.
Single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for understanding cellular heterogeneity and function. However the choice of sample multiplexing reagents can impact data quality ...and experimental outcomes. In this study, we compared various multiplexing reagents, including MULTI-Seq, Hashtag antibody, and CellPlex, across diverse sample types such as human peripheral blood mononuclear cells (PBMCs), mouse embryonic brain and patient-derived xenografts (PDXs). We found that all multiplexing reagents worked well in cell types robust to ex vivo manipulation but suffered from signal-to-noise issues in more delicate sample types. We compared multiple demultiplexing algorithms which differed in performance depending on data quality. We find that minor improvements to laboratory workflows such as titration and rapid processing are critical to optimal performance. We also compared the performance of fixed scRNA-Seq kits and highlight the advantages of the Parse Biosciences kit for fragile samples. Highly multiplexed scRNA-Seq experiments require more sequencing resources, therefore we evaluated CRISPR-based destruction of non-informative genes to enhance sequencing value. Our comprehensive analysis provides insights into the selection of appropriate sample multiplexing reagents and protocols for scRNA-Seq experiments, facilitating more accurate and cost-effective studies.
•Comparison of sample multiplexing reagents for single-cell RNA-Seq.•Evaluation of commercial fixed single-cell RNA-Seq kits.•Evaluation of CRISPR depletion of abundant transcripts.•Guidelines for implementation of cost saving measures for single-cell RNA-Seq.
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