4 research outputs found
Self-renewing Pten ---TP53 --- protospheres produce metastatic adenocarcinoma cell lines with multipotent progenitor activity
No full textProstate cancers of luminal adenocarcinoma histology display a range of clinical behaviors. Although most prostate cancers are slow-growing and indolent, a proportion is aggressive, developing metastasis and resistance to androgen deprivation treatment. One hypothesis is that a portion of aggressive cancers initiate from stem-like, androgen-independent tumor-propagating cells. Here we demonstrate the in vitro creation of a mouse cell line, selected for growth as self-renewing stem-progenitor cells, which manifests many in vivo properties of aggressive prostate cancer. Normal mouse prostate epithelium containing floxed Pten and TP53 alleles was subjected to CRE-mediated deletion in vitro followed by serial propagation as protospheres. A polyclonal cell line was established from dissociated protospheres and subsequently a clonal daughter line was derived. Both lines demonstrate a mature luminal phenotype in vitro. The established lines contain a stable minor population of progenitor cells with protosphere-forming ability and multi-lineage differentiation capacity. Both lines formed orthotopic adenocarcinoma tumors with metastatic potential to lung. Intracardiac inoculation resulted in brain and lung metastasis, while intra-tibial injection induced osteoblastic bone formation, recapitulating the bone metastatic phenotype of human prostate cancer. The cells showed androgen receptor dependent growth in vitro. Importantly, in vivo, the deprivation of androgens from established orthotopic tumors resulted in tumor regression and eventually castration-resistant growth. These data suggest that transformed prostate progenitor cells preferentially differentiate toward luminal cells and recapitulate many characteristics of the human disease.Abou-Kheir WG, 2010, STEM CELLS, V28, P2129, DOI 10.1002-stem.538; Agell L, 2008, MODERN PATHOL, V21, P1470, DOI 10.1038-modpathol.2008.145; Chen ZB, 2005, NATURE, V436, P725, DOI 10.1038-nature03918; Clevers H, 2011, NAT MED, V17, P313, DOI 10.1038-nm.2304; Goldstein AS, 2010, SCIENCE, V329, P568, DOI 10.1126-science.1189992; Goldstein AS, 2010, MOL ONCOL, V4, P385, DOI 10.1016-j.molonc.2010.06.009; Harris WP, 2009, NAT CLIN PRACT UROL, V6, P76, DOI 10.1038-ncpuro1296; Li H, 2008, CANCER RES, V68, P1820, DOI 10.1158-0008-5472.CAN-07-5878; Lim E, 2009, NAT MED, V15, P907, DOI 10.1038-nm.2000; Logothetis CJ, 2005, NAT REV CANCER, V5, P21, DOI 10.1038-nrc1528; MARTIN PL, 2011, AM J PATHOLOGY; Mellado B, 2009, CLIN TRANSL ONCOL, V11, P5, DOI 10.1007-s12094-009-0304-3; Molyneux G, 2010, CELL STEM CELL, V7, P403, DOI 10.1016-j.stem.2010.07.010; Mulholland DJ, 2009, CANCER RES, V69, P8555, DOI 10.1158-0008-5472.CAN-08-4673; Patrawala L, 2007, CANCER RES, V67, P6796, DOI 10.1158-0008-5472.CAN-07-0490; Patrawala L, 2006, ONCOGENE, V25, P1696, DOI 10.1038-sj.onc.1209327; Rajasekhar VK, 2011, NAT COMMUN, V2, DOI 10.1038-ncomms1159; Reya T, 2001, NATURE, V414, P105, DOI 10.1038-35102167; Roudier MP, 2003, HUM PATHOL, V34, P646, DOI 10.1016-S0046-8177(03)00190-4; Roudier MP, 2008, J UROLOGY, V180, P1154, DOI 10.1016-j.juro.2008.04.140; Schlomm T, 2008, MODERN PATHOL, V21, P1371, DOI 10.1038-modpathol.2008.104; Sircar K, 2009, J PATHOL, V218, P505, DOI 10.1002-path.2559; Song MS, 2011, CELL, V144, P187, DOI 10.1016-j.cell.2010.12.020; Stambolic V, 1998, CELL, V95, P29, DOI 10.1016-S0092-8674(00)81780-8; Sun H, 1999, P NATL ACAD SCI USA, V96, P6199, DOI 10.1073-pnas.96.11.6199; Taylor BS, 2010, CANCER CELL, V18, P11, DOI 10.1016-j.ccr.2010.05.026; Visvader JE, 2009, GENE DEV, V23, P2563, DOI 10.1101-gad.1849509; Wang X, 2009, NATURE, V461, P495, DOI 10.1038-nature08361; Wang ZA, 2011, ONCOGENE, V30, P1261, DOI 10.1038-onc.2010.53013
Identification of new markers for the characterization and isolation of breast cancer stem cells
No full textBreast cancer is the first human carcinoma for which a putative cancer stem cell subpopulation (BCSC) has been isolated with the CD44+/CD24-/low phenotype. However, several studies have highlighted that CD44+/CD24-/low cannot be considered the universal marker phenotype of BCSCs. The aim of this thesis is to identify novel BCSCs markers. We focused our work on the basal-like breast cancers that seem to derive from a developmental stage of mammary epithelial cell that is different from the primitive stem cell, namely the luminal progenitor.
In the first part of the present thesis we have characterized breast cancer cell lines resembling different tumor molecular subtypes, by using polychromatic flow cytometry. These analyses led us to hypothesize that the basal-A cell line HCC1937 could contain a putative CD24+CD338+ CSC subpopulation. We have next found that CD338 overexpressing cells, namely CD338bright, overlap, almost completely, with the side population in the HCC1937 cell line. Since side population is a property of stem/progenitor cells, this result suggested a key role of CD338 in determining stem-like properties of HCC1937 cell line. To explore this hypothesis, we next isolated the putative BCSCs, on the base of CD24 and CD338 expression, and we further studied them to explore their stem-like and tumorigenic properties. We found that CD24hi cells display a higher mammosphere forming efficiency than CD24- cells and, among CD24hi cells, CD338 overexpressing cells are able to form mammospheres with higher efficiency than CD338- cells. This result confirmed a relevant role of CD338 in stem-like properties of HCC1937 cell line. Furthermore, the soft-agar colony assays revealed that CD338 positive cells are transformed in contrary to the negative ones. We next evaluated the tumorigenic potential of the different CD338 sorted subpopulations and we found that cells expressing CD338 at an intermediate level, namely CD338low, were the most tumorigenic ones. Furthermore, the analysis of CD338bright and CD338low sorted sub-populations, after their growth in culture for several weeks, revealed that CD338bright population is able to divide asymmetrically giving rise to its CD338low progeny which, in contrast, is not able to divide asymmetrically. This result suggested that CD338bright population could constitute the most immature population and its CD338low progeny the more differentiated progenitor cells. Taken together, these results strongly support the presence in the HCC1937 cell line of a putative BCSC subpopulation overexpressing CD338 and bearing the phenotype CD44+CD24+CD338bright, i.e. an antigenic combination different from the classical CD44+/CD24-/low.
Concurrently to the study of breast cancer cell lines, a complementary work was carried out to standardize the flow-cytomertic methods to study primary cultures established from human breast cancer tissues. This work will be subsequently used to isolate and study the CD44+CD24+CD338bright CSC population from human breast cancer tissues. In this study, we have also tried to determine whether patient-derived breast cancer cell cultures maintain the cellular heterogeneity of primary tissues and may therefore be used for in vitro modeling of breast cancer subtypes. To this aim, we used a much larger vocabulary of surface markers than those used in previous studies to characterize primary cultures. Most of surface antigens analyzed were heterogeneously expressed. On the other hand, breast cancer cell cultures displayed concomitant high expression of the basal marker CD10/CALLA and low expression of CD326/EpCAM. Furthermore, we found that they express CK5, SMA and vimentin, and are weakly positive for CK19 and CK18-negative. Our results, while confirming that in vitro culturing of breast cancer cells reduces luminal lineage-type of cells, indicate the increased propensity for the selection of myoepithelial/basal breast cancer cells. Furthermore, we have demonstrated that breast cancer cell cultures preserve inter-tumor heterogeneity and express stem/progenitor markers that can be identified, quantified and categorized by flow cytometry
Accuracy of the Wound Healing Questionnaire in the diagnosis of surgical-site infection after abdominal surgery in low- and middle-income countries
No full textIntroductionTelemedicine is being adopted for postoperative surveillance but requires evaluation for efficacy. This study tested a telephone Wound Healing Questionnaire (WHQ) to diagnose surgical site infection (SSI) after abdominal surgery in low- and middle-income countries.MethodA multi-centre, international, prospective study was embedded in the FALCON trial; a factorial RCT testing measures to reduce SSI in seven low- and middle-income countries (NCT03700749). It was conducted according to a pre-registered protocol (SWAT126) and reported according to STARD guidelines. The reference test was in-person review by a trained clinician at 30 postoperative days according to US Centres for Disease Control criteria. The index test was telephone administration of an adapted WHQ at 27 to 30 postoperative days by a researcher blinded to the outcome of in-person review. The sum of item response scores generated an overall score between 0 and 29. The primary outcome was the diagnostic accuracy of the WHQ, defined as the proportion of SSI correctly identified by the telephone WHQ, and summarized using the area under the receiving operator characteristic curve (AUROC) and diagnostic test accuracy statistics.ResultsPatients were included from three upper-middle income (396 patients, 13 hospitals), three lower-middle income (746 patients, 19 hospitals), and one low-income country (54 patients, 4 hospitals). 90.3% (1088 of 1196) patients were successfully contacted. Those with non-midline incisions (adjusted odds ratio: 0.36, 95% c.i. 0.17 to 0.73, P=0.005) or a confirmed diagnosis of SSI on in-person assessment (odds ratio: 0.42, 95% c.i. 0.20 to 0.92, P=0.006) were harder to reach. The questionnaire correctly discriminated between most patients with and without SSI (AUROC 0.869, 95% c.i. 0.824 to 0.914), which was consistent across subgroups. A representative cut-off score of ≥4 displayed a sensitivity of 0.701 (0.610-0.792), specificity of 0.911 (0.878-0.943), positive predictive value of 0.723 (0.633-0.814) and negative predictive value of 0.901 (0.867-0.935).ConclusionSSI can be diagnosed using a telephone questionnaire (obviating in-person assessment) in low resource settings
Adaptation of the Wound Healing Questionnaire universal-reporter outcome measure for use in global surgery trials (TALON-1 study): mixed-methods study and Rasch analysis
No full textBackground: The Bluebelle Wound Healing Questionnaire (WHQ) is a universal-reporter outcome measure developed in the UK for remote detection of surgical-site infection after abdominal surgery. This study aimed to explore cross-cultural equivalence, acceptability, and content validity of the WHQ for use across low- and middle-income countries, and to make recommendations for its adaptation. Methods: This was a mixed-methods study within a trial (SWAT) embedded in an international randomized trial, conducted according to best practice guidelines, and co-produced with community and patient partners (TALON-1). Structured interviews and focus groups were used to gather data regarding cross-cultural, cross-contextual equivalence of the individual items and scale, and conduct a translatability assessment. Translation was completed into five languages in accordance with Mapi recommendations. Next, data from a prospective cohort (SWAT) were interpreted using Rasch analysis to explore scaling and measurement properties of the WHQ. Finally, qualitative and quantitative data were triangulated using a modified, exploratory, instrumental design model. Results: In the qualitative phase, 10 structured interviews and six focus groups took place with a total of 47 investigators across six countries. Themes related to comprehension, response mapping, retrieval, and judgement were identified with rich cross-cultural insights. In the quantitative phase, an exploratory Rasch model was fitted to data from 537 patients (369 excluding extremes). Owing to the number of extreme (floor) values, the overall level of power was low. The single WHQ scale satisfied tests of unidimensionality, indicating validity of the ordinal total WHQ score. There was significant overall model misfit of five items (5, 9, 14, 15, 16) and local dependency in 11 item pairs. The person separation index was estimated as 0.48 suggesting weak discrimination between classes, whereas Cronbach's α was high at 0.86. Triangulation of qualitative data with the Rasch analysis supported recommendations for cross-cultural adaptation of the WHQ items 1 (redness), 3 (clear fluid), 7 (deep wound opening), 10 (pain), 11 (fever), 15 (antibiotics), 16 (debridement), 18 (drainage), and 19 (reoperation). Changes to three item response categories (1, not at all; 2, a little; 3, a lot) were adopted for symptom items 1 to 10, and two categories (0, no; 1, yes) for item 11 (fever). Conclusion: This study made recommendations for cross-cultural adaptation of the WHQ for use in global surgical research and practice, using co-produced mixed-methods data from three continents. Translations are now available for implementation into remote wound assessment pathways
