Katherine Spence

A Detailed Mammosphere Assay Protocol for the Quantification of Breast Stem Cell Activity

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.

Wnt Pathway Activity in Breast Cancer Sub-Types and Stem-Like Cells

Introduction Wnt signalling has been implicated in stem cell regulation however its role in breast cancer stem cell regulation remains unclear. Methods We used a panel of normal and breast cancer cell lines to assess Wnt pathway gene and protein expression, and for the investigation of Wnt signalling within stem cell-enriched populations, mRNA and protein expression was analysed after the selection of anoikis-resistant cells. Finally, cell lines and patient-derived samples were used to investigate Wnt pathway effects on stem cell activity in vitro. Results Wnt pathway signalling increased in cancer compared to normal breast and in both cell lines and patient samples, expression of Wnt pathway genes correlated with estrogen receptor (ER) expression. Furthermore, specific Wnt pathway genes were predictive for recurrence within subtypes of breast cancer. Canonical Wnt pathway genes were increased in breast cancer stem cell-enriched populations in comparison to normal breast stem cell-enriched populations. Furthermore in cell lines, the ligand Wnt3a increased whilst the inhibitor DKK1 reduced mammosphere formation with the greatest inhibitory effects observed in ER+ve breast cancer cell lines. In patient-derived metastatic breast cancer samples, only ER-ve mammospheres were responsive to the ligand Wnt3a. However, the inhibitor DKK1 efficiently inhibited both ER+ve and ER-ve breast cancer but not normal mammosphere formation, suggesting that the Wnt pathway is aberrantly activated in breast cancer mammospheres. Conclusions Collectively, these data highlight differential Wnt signalling in breast cancer subtypes and activity in patient-derived metastatic cancer stem-like cells indicating a potential for Wnt-targeted treatment in breast cancers.

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

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.

Differential influence of TGFβ1 and TGFβ3 isoforms on cell cycle kinetics and postirradiation recovery of normal and malignant colorectal epithelial cells

PURPOSE A clonogenic assay was used to determine the effects of the growth factors TGFbeta1 and TGFbeta3 on the radiation responses of a normal rat epithelial cell line (IEC6) and a human colonic carcinoma epithelial cell line (Widr). METHODS AND MATERIALS The radiation sensitivity and ability to recover from potentially lethal damage (PLD), of preconfluent monolayer cultures, was assessed in the presence of the growth factors for 24 h prior to, during, and after irradiation. RESULTS The surviving fractions of both cell lines assessed immediately following irradiation were unaffected by TGFbeta1 or TGFbeta3. However, TGFbeta3 (but not TGFbeta1) significantly reduced the amount of PLD recovery in the Widr cells (but not in the IEC6 cells). This was associated with a reduction in the shoulder region of the survival curve, rather than a change in slope. A comparative analysis of the effects of TGFbetas 1 and 3 on cell cycle events in the two cell lines demonstrated significantly more Widr cells in the S phase, in the presence of TGFbeta3 only, compared to the controls. This remained constant both before and immediately following irradiation. In the IEC6 cell line TGFbeta3 produced an increase in the numbers of G1 phase cells, characteristic of a G1 arrest. CONCLUSION It seems likely that TGFbeta3-induced radiosensitisation in Widr cells, 6 h after a single dose of irradiation, is related to its effects on cell cycle events such that the failure of these cells to arrest in G1, either before or after irradiation, results in significantly reduced recovery from DNA damage. This, however, may not be the only mechanism by which this growth factor produces this effect. Indeed, it will also be necessary to investigate these effects in in vivo models and to determine the response to fractionated irradiation before the potential therapeutic benefit of both the differential effects observed between the two TGFbeta isoforms and also between the malignant and normal cell lines can be fully assessed.

Lack of caveolin-1 (P132L) somatic mutations in breast cancer

We were interested to read Patani et al.’s article [1] where they reported that they did not detect the p.P132L somatic mutation in CAV1 in a series of 240 breast tumors. We have also undertaken a study to determine the prevalence of the CAV1 p.P132L mutation in a series of 537 frozen tissue samples collected at the time of primary surgery from women with stages I, II, or III breast cancer from Manchester, UK between 1989 and 1998 (Table 1). Clinical data was collected by a comprehensive retrospective case note review to supplement prospectively collected data. The study was approved by local research ethics committee (08/H1006/77). Genomic DNA was extracted from fresh-frozen tissues using the EZ1 DNA tissue kit on EZ1 biorobot (Qiagen, Valencia, CA). CAV1 p.P132L mutation status was not identified by Pyrosequencing (Qiagen) in genomic DNA extracted from 537 tumor samples (call rate: 99.3%) (for full method see Supplementary material). To confirm the absence of p.P132L, samples with the highest allele quantification were analyzed by both direct DNA sequencing and restriction fragment length polymorphism analysis with the Nla-III restriction endonuclease. No mutations were detected. Furthermore, to ensure that mutations were not present at a level below the threshold of test sensitivity, we used laser capture microscopy (LCM) in a subset of 15 specimens from patients theoretically enriched for p.P132L mutation (ER-positive and well-differentiated tumors) [2]. DNA extracted from isolated tumor cells was sequenced in both orientations and no mutation was detected. The existence of CAV1 mutations in human breast cancers is controversial. In 2001 Hayashi et al. [3] reported that up to 16% of breast cancer tumor samples (mainly in ‘‘scirrhous’’ carcinomas) harbor a somatic mutation, p.P132L, in CAV1. Subsequent studies demonstrated that the p.P132L mutation was associated with ER-positive, but not ER-negative, breast tumors [4, 5]. However, other groups have not confirmed the existence of this somatic mutation in human breast cancers [6, 7]. Discordant results may reflect differences in patient populations, source of tumor materials and methods used [8]. DNA sequencing of formalin-fixed paraffin-embedded samples is fraught with difficulties, owing to the high prevalence of artefacts, with up to one artefactual mutation per 500 bases has been reported [9]. Importantly, in our study fresh-frozen samples were used whereas formalin-fixed paraffin-embedded samples were screened by groups where mutations were Electronic supplementary material The online version of this article (doi:10.1007/s10549-012-1981-0) contains supplementary material, which is available to authorized users.

Abstract PD2-02: SFX-01 targets Wnt signalling to inhibit stem-like cells in breast cancer patient-derived xenograft tumours

Background: SFX-01 is a novel therapeutic comprising synthetic sulforaphane (SFN) stabilised within a-cyclodextrin. Breast cancer stem-like cells (CSCs) have been identified in all molecular subtypes and are likely drivers of breast cancer metastasis and treatment resistance. We recently established that CSC activity in ER+ BC, represent a source of therapeutic resistance (Simoes et al, Cell Reports, 2015). Material and methods: We investigated SFX-01 effects on breast CSC activity using mammosphere formation efficiency (MFE) and aldehyde dehydrogenase (ALDH) activity using the ALDEFLUOR assay in patient samples and patient-derived xenograft (PDX) tumours. Cells from primary (n=12) and metastatic (n=15) samples were treated with SFX-01 (5 μM) or vehicle control.Using a 2 or 8 week in vivo treatment, early (HBCx34) and metastatic (BB3RC31) ER+ PDX tumours were treated with SFX-01 (300mg/Kg/day) alone or in combination with tamoxifen (TAM, 10 mg/kg/day) or fulvestrant (FULV, 200 mg/kg/week). Tumours were dissociated and MFE and ALDH activity assessed. Results: SFX-01 in vitro reduced MFE of both primary (0.19%±0.02 vs control 0.52%±0.06: p SFX-01 has been shown to be well tolerated in SAD and MAD studies in normal volunteers and clinical studies designed to test tolerability and efficacy in combination with the three major classes of endocrine therapy (AI, TAM and FULV) in advanced BC will begin in Q4 2016. Conclusions: Our data demonstrate the potential of SFX-01 for clinically meaningful improvements to endocrine therapy in ER+ breast cancer by reversing CSC mediated resistance. Citation Format: Howell SJ, Simoes BM, Alferez D, Eyre R, Spence K, Santiago-Gomez A, Sarmiento-Castro A, Tanaka I, Howat D, Clarke RB. SFX-01 targets Wnt signalling to inhibit stem-like cells in breast cancer patient-derived xenograft tumours [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD2-02.

Abstract P2-06-02: Breast cancer stem-like cell activity correlates with tumour progression to metastasis but not with clinical or tumour characteristics

Introduction: Breast cancers exhibit cellular heterogeneity, containing both stem-like and more differentiated cells. The activity of cancer stem cells (CSC) is likely to be dependent on the microenvironment or niche. Using 158 patient tumour samples, correlations between niche-independent breast CSC activity and clinical and tumour characteristics were tested. Methods: 104 early breast cancer surgical samples and 54 unrelated metastatic samples from pleural or ascitic fluid were harvested. To test CSC activity, isolated cells were grown in both primary (formation) and secondary (self-renewal) mammosphere (MS) culture. Tumour initiating activity was also tested by transplanting breast cancer fragments or cells into the sub-cutaneous flanks of NSG mice (n=84 early and n=10 metastatic). Results: No correlation was found between MS growth, MS formation (%), MS self-renewal (%) or in vivo tumour initiation and breast cancer sub-type, grade, node status or Nottingham prognostic index. 33% of the samples that formed MS in vitro initiated tumours in vivo while only 9% that failed to form MS initiated tumour growth. Metastatic compared to early BC samples grew MS more frequently (53/54 compared to 81/104), and had a higher primary MS formation efficiency (1% vs 0.6%; P Conclusions: In summary, niche-independent breast CSC activity measured in vitro by MS assay and in vivo by xenograft growth is not directly correlated with standard clinical parameters. However, both in vitro and in vivo CSC activity are increased in metastatic samples. These results suggest that breast CSC activity is independent of other prognostic indicators but may predict for poor outcome tumours. Relapse free survival data are maturing and will be presented with analysis of primary tumour ALDH1 expression. Citation Format: Sacha J Howell, Denis Alferez, Katherine Spence, Rachel Eyre, Fran Shaw, Bruno Simoes, Angelica Santiago-Gomez, Maria Bramley, Mohamed Absar, Zahida Saad, Sumohan Chatterjee, Cliona Kirwan, Ashu Gandhi, Anne C Armstrong, Andrew M Wardley, Gillian Farnie, Robert B Clarke. Breast cancer stem-like cell activity correlates with tumour progression to metastasis but not with clinical or tumour characteristics [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-06-02.