preparation-user-defined-peptide-gel-for-controlled-3d-culture-models

Preparation of a User-Defined Peptide Gel for Controlled 3D Culture Models of Cancer and Disease

There is a growing awareness that cells grown in 3D better model in vivo behavior than those grown in 2D. In this protocol, we describe a simple and ...

University of Manchester

Breast epithelial stem cells are thought to be the primary targets in the etiology of breast cancer. Since breast cancers mostly express estrogen and progesterone receptor (ERalpha and PR), we examined the biology of these ERalpha/PR-positive cells and their relationship to stem cells in normal human breast epithelium. We employed several complementary approaches to identify putative stem cell markers, to characterise an isolated stem cell population and to relate these to cells expressing the steroid receptors ERalpha and PR. Using DNA radiolabelling in human tissue implanted into athymic nude mice, a population of label-retaining cells were shown to be enriched for the putative stem cell markers p21(CIP1) and Msi-1, the human homolog of Drosophila Musashi. Steroid receptor-positive cells were found to co-express these stem cell markers together with cytokeratin 19, another putative stem cell marker in the breast. Human breast epithelial cells with Hoechst dye-effluxing "side population" (SP) properties characteristic of mammary stem cells in mice were demonstrated to be undifferentiated "intermediate" cells by lack of expression of myoepithelial and luminal apical membrane markers. These SP cells were 6-fold enriched for ERalpha-positive cells and expressed several fold higher levels of the ERalpha, p21(CIP1) and Msi1 genes than non-SP cells. In contrast to non-SP cells, SP cells formed branching structures in matrigel which included cells of both luminal and myoepithelial lineages. The data suggest a model where scattered steroid receptor-positive cells are stem cells that self-renew through asymmetric cell division and generate patches of transit amplifying and differentiated cells.

A putative human breast stem cell population is enriched for steroid receptor-positive cells.

&#x4E0A;&#x76AE;&#x6210;&#x9577;&#x56E0;&#x5B50;&#x53D7;&#x5BB9;&#x4F53; (EGFR) &#x3068; Notch &#x7D4C;&#x8DEF;&#x901A;&#x5E38;&#x80F8;&#x306E;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x81EA;&#x5DF1;&#x8907;&#x88FD;&#x306B;&#x95A2;&#x4E0E;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x81B5;&#x4E0A;&#x76AE;&#x5185;&#x764C; (DCIS) &#x4E73;&#x623F;&#x306E;&#x3053;&#x308C;&#x3089;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x7D4C;&#x8DEF;&#x306E;&#x95A2;&#x4E0E;&#x3092;&#x691C;&#x8A0E;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;

&#x30D7;&#x30E9;&#x30A4;&#x30DE;&#x30EA;&#x6027;&#x81B5;&#x7BA1;&#x4E0A;&#x76AE;&#x5185;&#x306E;&#x65B0;&#x898F;&#x7D30;&#x80DE;&#x57F9;&#x990A;&#x6280;&#x8853;: &#x30CE;&#x30C3;&#x30C1;&#x3068;&#x4E0A;&#x76AE;&#x6210;&#x9577;&#x56E0;&#x5B50;&#x53D7;&#x5BB9;&#x4F53;&#x306E;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x7D4C;&#x8DEF;&#x306E;&#x5F79;&#x5272;&#x3002;

&#xD45C; &#xD53C; &#xC131;&#xC7A5; &#xC778;&#xC790; &#xC218;&#xC6A9; &#xCCB4; (EGFR) &#xBC0F; &#xB178;&#xCE58; &#xC2E0;&#xD638; &#xACBD;&#xB85C; &#xC815;&#xC0C1;&#xC801;&#xC778; &#xC720; &#xBC29; &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xC758; &#xC790;&#xAE30; &#xAC31;&#xC2E0;&#xC5D0; &#xC5F0;&#xB8E8; &#xB418;&#xC5B4;. &#xC6B0;&#xB9AC;&#xB294; &#xC774;&#xB7EC;&#xD55C; &#xC2E0;&#xD638; &#xACBD;&#xB85C; ductal &#xC554; &#xC81C;&#xC790;&#xB9AC; (DCIS) &#xAC00;&#xC2B4;&#xC5D0; &#xCC38;&#xC5EC; &#xC870;&#xC0AC;.

&#xAE30;&#xBCF8; ductal &#xC554; &#xC81C;&#xC790;&#xB9AC;&#xB97C; &#xC704;&#xD55C; &#xC18C;&#xC124; &#xC140; &#xBB38;&#xD654; &#xAE30;&#xC220;: &#xB178;&#xCE58;&#xC640; &#xC2E0;&#xD638; &#xACBD;&#xB85C; &#xD45C; &#xD53C; &#xC131;&#xC7A5; &#xC778;&#xC790; &#xC218;&#xC6A9; &#xCCB4;&#xC758; &#xC5ED;&#xD560;.

El receptor del factor de crecimiento epid&eacute;rmico (EGFR) y muesca v&iacute;as de se&ntilde;alizaci&oacute;n han sido implicados en la renovaci&oacute;n de c&eacute;lulas de mama normales. Investigamos la participaci&oacute;n de estas v&iacute;as de se&ntilde;alizaci&oacute;n en el carcinoma ductal in situ (CDIS) del pecho.

T&eacute;cnica de cultivo celular de novela para primario carcinoma ductal in situ: papel de Notch y del receptor del factor de crecimiento epid&eacute;rmico v&iacute;as de se&ntilde;alizaci&oacute;n.

The epidermal growth factor receptor (EGFR) and Notch signaling pathways have been implicated in self-renewal of normal breast stem cells. We investigated the involvement of these signaling pathways in ductal carcinoma in situ (DCIS) of the breast.

Novel cell culture technique for primary ductal carcinoma in situ: role of Notch and epidermal growth factor receptor signaling pathways.

Le r&eacute;cepteur du facteur de croissance &eacute;pidermique (EGFR) et l&#39;encoche, voies de signalisation ont &eacute;t&eacute; impliqu&eacute;s dans l&#39;autorenouvellement des cellules souches normales du sein. Nous avons &eacute;tudi&eacute; l&#39;implication de ces voies de signalisation dans le carcinome canalaire in situ (CCIS) du sein.

Technique de culture cellulaire roman pour primaire carcinome canalaire in situ : r&ocirc;le de l&#39;encoche et epidermal growth factor receptor signaling pathways.

Die epidermalen Wachstumsfaktor-Rezeptor (EGFR) und Notch Signalisierung Wege haben in Selbsterneuerung von Stammzellen der normalen Brust verwickelt. Wir untersuchten die Einbeziehung dieser Signalwege in duktale Carcinoma in situ (DCIS) der Brust.

Neuartige Zelle Kultur Technik f&uuml;r prim&auml;re duktale Carcinoma in situ: Rolle der Kerbe und epidermalen Wachstumsfaktor-Rezeptor Signalgebung Wege.

Il recettore del fattore di crescita epidermico (EGFR) e tacca vie di segnalazione sono stati implicati nell&#39;auto-rinnovamento delle cellule staminali normali del seno. Abbiamo studiato il coinvolgimento di queste vie di segnalazione nel carcinoma duttale in situ (DCIS) del seno.

Tecnica di coltura cellulare romanzo per primario carcinoma duttale in situ: ruolo della tacca ed epidermal growth factor receptor signaling pathways.

&#x8868;&#x76AE;&#x751F;&#x957F;&#x56E0;&#x5B50;&#x53D7;&#x4F53; (EGFR) &#x548C; Notch &#x901A;&#x8DEF;&#x6709;&#x7275;&#x8FDE;&#x6B63;&#x5E38;&#x4E73;&#x817A;&#x5E72;&#x7EC6;&#x80DE;&#x7684;&#x81EA;&#x6211;&#x66F4;&#x65B0;&#x3002;&#x8FD9;&#x4E9B;&#x4FE1;&#x53F7;&#x4F20;&#x5BFC;&#x901A;&#x8DEF;&#x5728;&#x4E73;&#x817A;&#x5BFC;&#x7BA1;&#x539F;&#x4F4D;&#x764C; &#xFF08;DCIS&#xFF09; &#x7684;&#x4E73;&#x817A;&#x4E2D;&#x7684;&#x53C2;&#x4E0E;&#xFF0C;&#x6211;&#x4EEC;&#x5BF9;&#x6B64;&#x8FDB;&#x884C;&#x8C03;&#x67E5;&#x3002;

&#x539F;&#x53D1;&#x6027;&#x4E73;&#x817A;&#x5BFC;&#x7BA1;&#x539F;&#x4F4D;&#x764C;&#x4E2D;&#x7684;&#x65B0;&#x578B;&#x7EC6;&#x80DE;&#x57F9;&#x517B;&#x6280;&#x672F;&#xFF1A; &#x51F9;&#x69FD;&#x548C;&#x8868;&#x76AE;&#x751F;&#x957F;&#x56E0;&#x5B50;&#x53D7;&#x4F53;&#x4FE1;&#x53F7;&#x901A;&#x8DEF;&#x7684;&#x4F5C;&#x7528;&#x3002;

Since the discovery that neural tissue contains a population of stem cells that form neurospheres in vitro, sphere-forming assays have been adapted for use with a number of different tissue types for the quantification of stem cell activity and self-renewal. One tissue type widely used for stem cell investigations is mammary tissue, and the mammosphere assay has been used in both normal tissue and cancer. Although it is a relatively simple assay to learn, it can be difficult to master. There are methodological and analytical aspects to the assay which require careful consideration when interpreting the results. We describe here a detailed mammosphere assay protocol for the assessment of stem cell activity and self-renewal, and discuss how data generated by the assay can be analysed and interpreted.

A detailed mammosphere assay protocol for the quantification of breast stem cell activity.

Wnt signalling has been implicated in stem cell regulation however its role in breast cancer stem cell regulation remains unclear.

Wnt pathway activity in breast cancer sub-types and stem-like cells.

Therapy resistance is one of the major impediments to successful cancer treatment. In breast cancer, a small subpopulation of cells with stem cell features, named breast cancer stem cells (BCSC), is responsible for metastasis and recurrence of the tumor. BCSC have the unique ability to grow under non-adherent conditions in "mammospheres". To prevent breast cancer recurrence and metastasis it will be crucial to eradicate BCSC.We used shRNA genetic screening to identify genes that upon knockdown enhance mammosphere formation in breast cancer cells. By integration of these results with gene expression profiles of mammospheres and NOTCH-activated cells, we identified FOXO3A. Modulation of FOXO3A activity results in a change in mammosphere formation, expression of mammary stem cell markers and breast cancer initiating potential. Importantly, lack of FOXO3A expression in breast cancer patients is associated with increased recurrence rate. Our findings provide evidence for a role for FOXO3A downstream of NOTCH and AKT that may have implications for therapies targeting BCSCs. 

An integrated genomic approach identifies that the PI3K/AKT/FOXO pathway is involved in breast cancer tumor initiation.

Observational studies suggest weight loss and energy restriction reduce breast cancer risk. Intermittent energy restriction (IER) reduces weight to the same extent as, or more than equivalent continuous energy restriction (CER) but the effects of IER on normal breast tissue and systemic metabolism as indicators of breast cancer risk are unknown.

Intermittent energy restriction induces changes in breast gene expression and systemic metabolism.

Estrogen receptor (ER) positive breast cancer is frequently sensitive to endocrine therapy. Multiple mechanisms of endocrine therapy resistance have been identified, including cancer stem-like cell (CSC) activity. Here we investigate SFX-01, a stabilised formulation of sulforaphane (SFN), for its effects on breast CSC activity in ER+ preclinical models. SFX-01 reduced mammosphere formation efficiency (MFE) of ER+ primary and metastatic patient samples. Both tamoxifen and fulvestrant increased MFE and aldehyde dehydrogenase (ALDH) activity of patient-derived xenograft (PDX) tumors, which was reversed by combination with SFX-01. SFX-01 significantly reduced tumor-initiating cell frequency in secondary transplants and reduced the formation of spontaneous lung micrometastases by PDX tumors in mice. Mechanistically, we establish that both tamoxifen and fulvestrant induce STAT3 phosphorylation. SFX-01 suppressed phospho-STAT3 and SFN directly bound STAT3 in patient and PDX samples. Analysis of ALDH+ cells from endocrine-resistant patient samples revealed activation of STAT3 target genes MUC1 and OSMR, which were inhibited by SFX-01 in patient samples. Increased expression of these genes after 3 months' endocrine treatment of ER+ patients (n = 68) predicted poor prognosis. Our data establish the importance of STAT3 signaling in CSC-mediated resistance to endocrine therapy and the potential of SFX-01 for improving clinical outcomes in ER+ breast cancer.

Article	Year
A putative human breast stem cell population is enriched for steroid receptor-positive cells. Developmental biology\| PubMed ID: 15617686	2005
Novel cell culture technique for primary ductal carcinoma in situ: role of Notch and epidermal growth factor receptor signaling pathways. Journal of the National Cancer Institute\| PubMed ID: 17440163	2007
A detailed mammosphere assay protocol for the quantification of breast stem cell activity. Journal of mammary gland biology and neoplasia\| PubMed ID: 22665270	2012
Wnt pathway activity in breast cancer sub-types and stem-like cells. PloS one\| PubMed ID: 23861811	2013
An integrated genomic approach identifies that the PI3K/AKT/FOXO pathway is involved in breast cancer tumor initiation. Oncotarget\| PubMed ID: 26595803	2016
Intermittent energy restriction induces changes in breast gene expression and systemic metabolism. Breast cancer research : BCR\| PubMed ID: 27233359	2016
Targeting STAT3 signaling using stabilised sulforaphane (SFX-01) inhibits endocrine resistant stem-like cells in ER-positive breast cancer. Oncogene\| PubMed ID: 32472077	2020

Katherine Spence

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