Breast cancer is an umbrella term used to describe a collection of different diseases with broad inter- and intratumor heterogeneity. Understanding this variation is critical in order to develop, and ...precisely prescribe, new treatments. Changes in the lipid metabolism of cancerous cells can provide important indications as to the metabolic state of the cells but are difficult to investigate with conventional histological methods. Due to the introduction of new higher energy (40 kV) gas cluster ion beams (GCIBs), time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging is now capable of providing information on the distribution of hundreds of molecular species simultaneously on a cellular to subcellular scale. GCIB-ToF-SIMS was used to elucidate changes in lipid composition in nine breast cancer biopsy samples. Improved molecular signal generation by the GCIB produced location-specific information that revealed elevated levels of essential lipids to be related to inflammatory cells in the stroma, while cancerous areas were dominated by nonessential fatty acids and a variety of phosphatidylinositol species with further in-tumor variety arising from decreased desaturase activity. These changes in lipid composition due to different enzyme activity are seemingly independent of oxygen availability and can be linked to favorable cell membrane properties for either proliferation/invasion or drug resistance/survival
Abstract
Three-dimensional cell culture platforms based on decellularised patient-based microenvironments provide in vivo-like growth conditions allowing cancer cells to interact with intact ...structures and components of the surrounding tissue. A patient-derived scaffold (PDS) model was therefore evaluated as a testing platform for the endocrine therapies (Z)-4-Hydroxytamoxifen (4OHT) and fulvestrant as well as the CDK4/6-inhibitor palbociclib, monitoring the treatment responses in breast cancer cell lines MCF7 and T47D adapted to the patient-based microenvironments. MCF7 cells growing in PDSs showed increased resistance to 4OHT and fulvestrant treatment (100- and 20-fold) compared to 2D cultures. Quantitative PCR analyses of endocrine treated cancer cells in PDSs revealed upregulation of pluripotency markers further supported by increased self-renewal capacity in sphere formation assays. When comparing different 3D growth platforms including PDS, matrigel, gelatin sponges and 3D-printed hydrogels, 3D based cultures showed slightly varying responses to fulvestrant and palbociclib whereas PDS and matrigel cultures showed more similar gene expression profiles for 4OHT treatment compared to the other platforms. The results support that the PDS technique maximized to provide a multitude of smaller functional PDS replicates from each primary breast cancer, is an up-scalable patient-derived drug-testing platform available for gene expression profiling and downstream functional assays.
Breast cancer stem-like cells (CSC) are an important therapeutic target as they are predicted to be responsible for tumor initiation, maintenance, and metastases. Interleukin (IL)-8 is upregulated in ...breast cancer and is associated with poor prognosis. Breast cancer cell line studies indicate that IL-8 via its cognate receptors, CXCR1 and CXCR2, is important in regulating breast CSC activity. We investigated the role of IL-8 in the regulation of CSC activity using patient-derived breast cancers and determined the potential benefit of combining CXCR1/2 inhibition with HER2-targeted therapy.
CSC activity of metastatic and invasive human breast cancers (n = 19) was assessed ex vivo using the mammosphere colony-forming assay.
Metastatic fluid IL-8 level correlated directly with mammosphere formation (r = 0.652; P < 0.05; n = 10). Recombinant IL-8 directly increased mammosphere formation/self-renewal in metastatic and invasive breast cancers (n = 17). IL-8 induced activation of EGFR/HER2 and downstream signaling pathways and effects were abrogated by inhibition of SRC, EGFR/HER2, phosphoinositide 3-kinase (PI3K), or MEK. Furthermore, lapatinib, which targets EGFR/HER2, inhibited the mammosphere-promoting effect of IL-8 in both HER2-positive and negative patient-derived cancers. CXCR1/2 inhibition also blocked the effect of IL-8 on mammosphere formation and added to the efficacy of lapatinib in HER2-positive cancers.
These studies establish a role for IL-8 in the regulation of patient-derived breast CSC activity and show that IL-8/CXCR1/2 signaling is partly mediated via a novel SRC and EGFR/HER2-dependent pathway. Combining CXCR1/2 inhibitors with current HER2-targeted therapies has potential as an effective therapeutic strategy to reduce CSC activity in breast cancer and improve the survival of HER2-positive patients.
The role of cancer-associated fibroblasts (CAFs) during tumour progression is obscured by the inherently complex, heterotypic nature of fibroblast cells and behaviours in various subtypes of ...malignancies. Therefore, we sought to identify distinct fibroblast subpopulations at the single-cell level.
Using single-cell quantitative PCR as a powerful tool to study heterogeneity and rare cell events, in a high-throughput manner a panel of gene targets are run simultaneously on transcripts isolated from single cells obtained by fluorescence-activated cell sort. Assessment of cells with stem-like characteristics was attained by anchorage-independent, anoikis-resistant culture.
Single-cell analysis of fibroblasts and their tumour-activated counterparts demonstrated molecularly distinct cell types defined by differential expression of characteristic mesenchymal and fibroblast activation markers. Identified subpopulations presented overlapping gene expression patterns indicating transitional molecular states during fibroblast differentiation. Using single-cell resolution data we generated a molecular differentiation model which enabled the classification of patient-derived fibroblasts, validating our modelling approach. Remarkably, a subset of fibroblasts displayed expression of pluripotency markers, which was enriched for in non-adherent conditions. Yet the ability to form single-cell derived spheres was generally reduced in CAFs and upon fibroblast activation through TGFβ1 ligand and cancer cell-secreted factors. Hence, our data imply the existence of putative stem/progenitor cells as a physiological feature of undifferentiated fibroblasts.
Within this comprehensive study we have identified distinct and intersecting molecular profiles defining fibroblast activation states and propose that underlying cellular heterogeneity, fibroblasts are hierarchically organized. Understanding the molecular make-up of cellular organization and differentiation routes will facilitate the discovery of more specific markers for stromal subtypes and targets for anti-stromal therapies.
Cancer progression is influenced by genetic aberrations in the cancer cell population as well as by other factors including the microenvironment present within a tumour. Direct interactions between ...various cell types as well as cellular signalling via secreted cytokines can drive key tumourigenic properties associated with disease progression and treatment resistance. Also, cancer stem cell functions are influenced by the microenvironment. This challenging subset of cells has been linked to malignant properties. Within a screen, using in vivo like growth conditions, we identified progranulin as a highly secreted cytokine affecting cancer stem cells in breast cancer. This cytokine is known to play a role in numerous biological and tumour-related processes including therapy resistance in a range of cancer types.
Different in vitro and in vivo relevant conditions were used to validate breast cancer stem cell expansion mediated by progranulin and its receptor sortilin. Small interfering ribonucleic acid (siRNA) and pharmacological inhibition of sortilin were used to elucidate the role of sortilin as a functional receptor during progranulin-induced breast cancer stem cell propagation, both in vitro and in vivo, using breast cancer xenograft models. In addition, single-cell gene expression profiling as well as a Sox2 reporter breast cancer cell line were used to validate the role of dedifferentiation mediated by progranulin.
In various in vivo-like screening assays, progranulin was identified as a potent cancer stem cell activator, highly secreted in ERα-negative breast cancer as well as in ERα-positive breast cancer under hypoxic adaptation. Progranulin exposure caused dedifferentiation as well as increased proliferation of the cancer stem cell pool, a process that was shown to be dependent on its receptor sortilin. Subcutaneous injections of progranulin or its active domain (GRN A) induced lung metastases in breast cancer xenograft models, supporting a major role for progranulin in cancer progression. Importantly, an orally bioavailable small molecule (AF38469) targeting sortilin, blocked GRN A-induced lung metastases and prevented cancer cell infiltration of the skin.
The collective results suggest that sortilin targeting represents a potential novel breast cancer therapy approach inhibiting tumour progression driven by secretion and microenvironmental influences.
The definitive characterization of common cancer stem cell (CSCs) subpopulations in breast cancer subtypes with distinct genotypic and phenotypic features remains an ongoing challenge. In this study, ...we have used a non-biased genome wide screening approach to identify transcriptional networks that may be specific to the CSC subpopulations in both luminal and basal breast cancer subtypes. In depth studies of three CSC-enriched breast cancer cell lines representing various subtypes of breast cancer revealed a striking hyperactivation of the mevalonate metabolic pathway in comparison to control cells. The upregulation of metabolic networks is a key feature of tumour cells securing growth and proliferative capabilities and dysregulated mevalonate metabolism has been associated with tumour malignancy and cellular transformation in breast cancer. Furthermore, accumulating evidence suggests that Simvastatin therapy, a mevalonate pathway inhibitor, could affect breast cancer progression and reduce breast cancer recurrence. When detailing the mevalonate pathway in breast cancer using a single-cell qPCR, we identified the mevalonate precursor enzyme, HMGCS1, as a specific marker of CSC-enriched subpopulations within both luminal and basal tumour subtypes. Down-regulation of HMGCS1 also decreased the CSC fraction and function in various model systems, suggesting that HMGCS1 is essential for CSC-activities in breast cancer in general. These data was supported by strong associations between HMGCS1 expression and aggressive features, such as high tumour grade, p53 mutations as well as ER-negativity in lymph node positive breast cancer. Importantly, loss of HMGCS1 also had a much more pronounced effect on CSC-activities compared to treatment with standard doses of Simvastatin. Taken together, this study highlights HMGCS1 as a potential gatekeeper for dysregulated mevalonate metabolism important for CSC-features in both luminal and basal breast cancer subtypes. Pharmacological inhibition of HMGCS1 could therefore be a superior novel treatment approach for breast cancer patients via additional CSC blocking functions.
Myxoid liposarcoma (MLS) shows extensive intratumoural heterogeneity with distinct subpopulations of tumour cells. Despite improved survival of MLS patients, existing therapies have shortcomings as ...they fail to target all tumour cells. The nature of chemotherapy‐resistant cells in MLS remains unknown. Here, we show that MLS cell lines contained subpopulations of cells that can form spheres, efflux Hoechst dye and resist doxorubicin, all properties attributed to cancer stem cells (CSCs). By single‐cell gene expression, western blot, phospho‐kinase array, immunoprecipitation, immunohistochemistry, flow cytometry and microarray analysis we showed that a subset of MLS cells expressed JAK–STAT genes with active signalling. JAK1/2 inhibition via ruxolitinib decreased, while stimulation with LIF increased, phosphorylation of STAT3 and the number of cells with CSC properties indicating that JAK–STAT signalling controlled the number of cells with CSC features. We also show that phosphorylated STAT3 interacted with the SWI/SNF complex. We conclude that MLS contains JAK–STAT‐regulated subpopulations of cells with CSC features. Combined doxorubicin and ruxolitinib treatment targeted both proliferating cells as well as cells with CSC features, providing new means to circumvent chemotherapy resistance in treatment of MLS patients.
What's new?
Despite improved survival of patients, existing therapies for Myxoid liposarcoma (MLS) present shortcomings as they fail to target all tumour cells. The nature of chemotherapy‐resistant cells in MLS remains unknown, however. Here, the authors show that myxoid liposarcomas are heterogeneous and contain subpopulations of cells with stem cell properties, including chemotherapy resistance. Moreover, JAK‐STAT signalling is active in MLS and regulates the size of the cancer stem cells‐like subpopulation via the SWI/SNF complex. The results shed light on the mechanisms of therapy resistance in MLS and point to JAK‐STAT inhibitors as a new avenue for targeted MLS therapies.
Colorectal cancer is a commonly diagnosed cancer worldwide. Unfortunately, many patients do not respond to standard chemotherapy treatments and develop disease relapse and metastases. Besides cancer ...cell specific genetic changes, heterogeneity in the tumor microenvironment contribute to the clinical presentation of the disease and can potentially also influence drug resistance. By using a recently developed patient-derived scaffold method monitoring how a standardized reporter cancer cell line adapts to various microenvironments treated with chemotherapy, we wanted to clarify how individual patient specific microenvironments influence the chemotherapy response in colorectal cancer.
Surgically resected colorectal cancer specimens from 89 patients were decellularized to produce patient-derived scaffold, which were seeded with HT29 cells, cultured for 3 weeks, and treated with 5-fluorouracil. Gene expression changes of adapted and treated HT29 cells were monitored by qPCR and compared with clinical parameters including disease-free survival.
The effects of 5-fluorouracil treatment varied between different patient-derived scaffold, but generally induced a reduced expression of proliferation genes and increased expression of pluripotency and epithelial-to-mesenchymal transition genes. Interestingly, patient-derived scaffold cultures obtained from patients with disease recurrences showed a significantly less pronounced anti-proliferative effect of 5-fluorouracil and more pronounced increase of pluripotency, with MKI67 and POU5F1 being among the most significant genes linked to disease relapse in colorectal cancer.
Colorectal patient-derived scaffold can decode clinically relevant tumor microenvironmental influence of 5-fluorouracil treatment effects opening up for optimized precision medicine in colorectal cancer treatment.
Cyclin D1, Id1 and EMT in breast cancer Tobin, Nicholas P; Sims, Andrew H; Lundgren, Katja L ...
BMC cancer,
09/2011, Letnik:
11, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Cyclin D1 is a well-characterised cell cycle regulator with established oncogenic capabilities. Despite these properties, studies report contrasting links to tumour aggressiveness. It has previously ...been shown that silencing cyclin D1 increases the migratory capacity of MDA-MB-231 breast cancer cells with concomitant increase in 'inhibitor of differentiation 1' (ID1) gene expression. Id1 is known to be associated with more invasive features of cancer and with the epithelial-mesenchymal transition (EMT). Here, we sought to determine if the increase in cell motility following cyclin D1 silencing was mediated by Id1 and enhanced EMT-features. To further substantiate these findings we aimed to delineate the link between CCND1, ID1 and EMT, as well as clinical properties in primary breast cancer.
Protein and gene expression of ID1, CCND1 and EMT markers were determined in MDA-MB-231 and ZR75 cells by western blot and qPCR. Cell migration and promoter occupancy were monitored by transwell and ChIP assays, respectively. Gene expression was analysed from publicly available datasets.
The increase in cell migration following cyclin D1 silencing in MDA-MB-231 cells was abolished by Id1 siRNA treatment and we observed cyclin D1 occupancy of the Id1 promoter region. Moreover, ID1 and SNAI2 gene expression was increased following cyclin D1 knock-down, an effect reversed with Id1 siRNA treatment. Similar migratory and SNAI2 increases were noted for the ER-positive ZR75-1 cell line, but in an Id1-independent manner. In a meta-analysis of 1107 breast cancer samples, CCND1low/ID1high tumours displayed increased expression of EMT markers and were associated with reduced recurrence free survival. Finally, a greater percentage of CCND1low/ID1high tumours were found in the EMT-like 'claudin-low' subtype of breast cancer than in other subtypes.
These results indicate that increased migration of MDA-MB-231 cells following cyclin D1 silencing can be mediated by Id1 and is linked to an increase in EMT markers. Moreover, we have confirmed a relationship between cyclin D1, Id1 and EMT in primary breast cancer, supporting our in vitro findings that low cyclin D1 expression can be linked to aggressive features in subgroups of breast cancer.
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