MFUM-BrTNBC-1, a Newly Established Patient-Derived Triple-Negative Breast Cancer Cell Line: Molecular Characterisation, Genetic Stability, and Comprehensive Comparison with Commercial Breast Cancer Cell Lines

Triple-negative breast cancer (TNBC) is a breast cancer (BC) subtype that accounts for approximately 15–20% of all BC cases. Cancer cell lines (CLs) provide an efficient way to model the disease. We have recently isolated a patient-derived triple-negative BC CL MFUM-BrTNBC-1 and performed a detailed morphological and molecular characterisation and a comprehensive comparison with three commercial BC CLs (MCF-7, MDA-MB-231, MDA-MB-453). Light and fluorescence microscopy were used for morphological studies; immunocytochemical staining for hormone receptor, p53 and Ki67 status; RNA sequencing, qRT-PCR and STR analysis for molecular characterisation; and biomedical image analysis for comparative phenotypical analysis. The patient tissue-derived MFUM-BrTNBC-1 maintained the primary triple-negative receptor status. STR analysis showed a stable and unique STR profile up to the 6th passage. MFUM-BrTNBC-1 expressed EMT transition markers and displayed changes in several cancer-related pathways (MAPK, Wnt and PI3K signalling; nucleotide excision repair; and SWI/SNF chromatin remodelling). Morphologically, MFUM-BrTNBC-1 differed from the commercial TNBC CL MDA-MB-231. The advantages of MFUM-BrTNBC-1 are its isolation from a primary tumour, rather than a metastatic site; good growth characteristics; phenotype identical to primary tissue; complete records of origin; a unique identifier; complete, unique STR profile; quantifiable morphological properties; and genetic stability up to (at least) the 6th passage.

Dekonvolution von Mischspuren nach vollständig kontinuierlichem Modell

Zusammenfassung Hintergrund Bei der Untersuchung von Mischspuren können stochastische Effekte die Beurteilung einer Spurenlegerschaft beeinträchtigen. Daher finden immer mehr softwarebasierte Lösungen Einzug in die Spurenuntersuchung, die durch Berücksichtigung biologischer Parameter eine Hilfestellung bei der Ableitung von Einzelprofilen bieten sollen. Im Rahmen der Studie wurde eine wissenschaftliche Validierung der Mischspurenanalyse-Software Genoproof® Mixture 4 (GPM4, Qualitype GmbH, Dresden, Deutschland) durchgeführt. Material und Methoden Die in unterschiedlichen Mischungsverhältnissen vorliegenden 2‑ und 3‑Personen-Mischspuren wurden künstlich unter Verwendung isolierter CD4+-Lymphozyten von 9 Spendenden erzeugt. Nach Erstellung der STR-Profile wurden die Mischspuren mittels GPM4 im Hinblick auf die Dekonvolution ausgewertet. Ergebnisse In den 2‑Personen-Mischspuren mit klarer Unterscheidung von Haupt- und Nebenkomponente wurde von der Software in der Großzahl der untersuchten STR-Systeme die richtige Genotypkonstellation (GTK) der Komponenten abgeleitet, oftmals mit einer Wichtung > 90 %. In den anteilsähnlicheren Mischspuren wurden zunehmend nichtzutreffende Allelableitungen beobachtet. Eine Abnahme der Performance in Bezug auf die Ableitung der richtigen GTK zeigten die 3‑Personen-Mischspuren. Faktoren wie Mischkomposition und Homo- und Heterozygotie in den genetischen Profilen hatten nachweislich einen Einfluss auf die Auftrennung der Mischspuren. Diskussion Mischspuren, die keine klare Unterscheidung von Haupt- und Nebenkomponente erlauben, stellen eine Schwierigkeit bei der Dekonvolution dar. In diesen Fällen ist eine Differenzierung der Peakhöhen detektierter Allele nur schwer möglich, da diese bei Anteilsgleichheit beider Komponenten eine komparable Intensität aufweisen. Ein deutlicher DNA-Mengen-Unterschied der Komponenten ist für die Berechnung von Vorteil.

Genetic analysis of father-daughter incest using multifaceted STR markers and study of inheritance pattern of alleles

Background: Two cases involving father-daughter incest, a rare report in the Indian population, have been analyzed in the current study. STR markers on both autosomal and sex chromosomes were employed to expound the cases. Objective: To confirm the identity of the fetus as a product of father-daughter incest and to study the inheritance pattern of alleles in such cases. Results: In both cases, the aborted fetus was found to be the product of an incestuous father-daughter relationship. The probability of paternity as well as maternity was found to be >99.9999% in both cases. Analysis of other paternity and forensic parameters also substantiated the inclusion of the alleged individuals. Father-daughter incest had a tremendous effect on the genome as evidenced from the dramatical decrease in unrelated alleles between father/child [16.66% (Case 1), 20% (Case 2)] and mother/child [26.66% (Case 1), 21.66% (Case 2)]. Genetic evidence also suggested an increased biallelic match i.e., 26.66% (Case 1) and 33.33% (Case 2) between mother and fetus which are at par/ above the normal siblings’ values i.e., 26.66%. Conclusion: A significant increase in the percentage of homozygous alleles (53.33% in both cases) was observed in the product of father-daughter incest. Both daughters share the same X chromosome from the father, which also suggested the case to be of father-daughter incest. Similarly, the same Y-STR profile between the male fetus and alleged father confirmed the correct pattern of inherit1ance of the Y chromosome in this case.

Progression and Differentiation of Alveolar Rhabdomyosarcoma Is Regulated by PAX7 Transcription Factor—Significance of Tumor Subclones

Rhabdomyosarcoma (RMS), is the most frequent soft tissue tumor in children that originates from disturbances in differentiation process. Mechanisms leading to the development of RMS are still poorly understood. Therefore, by analysis of two RMS RH30 cell line subclones, one subclone PAX7 negative, while the second one PAX7 positive, and comparison with other RMS cell lines we aimed at identifying new mechanisms crucial for RMS progression. RH30 subclones were characterized by the same STR profile, but different morphology, rate of proliferation, migration activity and chemotactic abilities in vitro, as well as differences in tumor morphology and growth in vivo. Our analysis indicated a different level of expression of adhesion molecules (e.g., from VLA and ICAM families), myogenic microRNAs, such as miR-206 and transcription factors, such as MYOD, MYOG, SIX1, and ID. Silencing of PAX7 transcription factor with siRNA confirmed the crucial role of PAX7 transcription factor in proliferation, differentiation and migration of RMS cells. To conclude, our results suggest that tumor cell lines with the same STR profile can produce subclones that differ in many features and indicate crucial roles of PAX7 and ID proteins in the development of RMS.

Influence of the degree of homozygosity of STR-loci on the fertility of thoroughbred horses

Purpose: to study the effect of the level of homozygosity and inbreeding on the fecundity of thoroughbred mares.Materials and methods. A total of 3662 heads of breeding sows from leading stud farms of the Russian Federation were selected for the analysis. The influence of the homozygosity level on 17 microsatellite DNA loci on the main indices of reproduction of thoroughbred mares, such as the safe yield of foals and the number of fetal years, was analyzed in the article. All mares in the experimental sample had at least three years of breeding use. Individual fecundity rates and the number of homozygous loci in the STR profile were calculated for each horse.Results. The highest live foal yields (75.92%) had sows with homozygosity levels of 62.78-69.02%, and the lowest foal yields (45.73%) were recorded in mares with the highest homozygosity levels (75.28-76.92%). The maximum foal yield (65.85%) was determined in sows with an inbreeding rate of 4.1% or more, with a productive longevity of 6.26 fetal years on average. Analysis of the data showed that the level of inbreeding had almost no effect on the yield of live foals (R=0.010 at P>0.05), but had a negative correlation with the number of fruiting years (R=-0.092 at P<0.005).Conclusion. Estimation of homozygosity level of thoroughbred horses is especially relevant, as this breed has been perfected by purebred breeding method only for more than thirty years. In the thoroughbred horse breed, it is necessary to create a system of maintaining heterozygosity as well as the diversity of genotypes through the organization of breeding work.

Evaluation and SNP typing of DNA from ultraviolet-irradiated human bloodstains using TaqMan assay

When detecting DNA profiles from forensic materials, it is pivotal to know the extent of degradation and which DNA marker can be genotyped. Ultraviolet (UV) is one of the common external factors that causes DNA damage, through which, an attempt to reveal cardinal genetic information can be made. In this study, after irradiation with three different UV wavelengths, UV-damaged DNA in the bloodstains was analyzed with long and short TaqMan assays using real-time PCR. In addition, both short tandem repeat (STR) profiles and single nucleotide polymorphisms (SNPs) from the damaged DNA at different stages of UV exposure were also assessed. With increasing in UV irradiation cycles, there was a delay of the amplification curves accompanied with a decrease in the DNA amounts collected. Despite the amplification of STR genotype was not altered after 75 cycles of UVC irradiation, all 12 SNP loci could still be detected. Furthermore, a short-assay line was detected in the absence of an amplification of the evaluation curve. The results indicate that, although the DNA template might not be useful and suitable for analysis of STR profile, this approach is of some values in detecting SNPs.

Genomic profiling of cell lines reveals hidden research bias and caveats

Cancer cell lines are extremely valuable tools for carcinoma research. Biodiversity of cell lines and continuous random mutation in cell lines during passage, however, might result in phenotypic inconsistency and lead to biased experimental conclusions. Using statistics based on known and inferred protein interaction networks, as well as public research literature database, our study shows that essential driver genotypes of cell lines might have hidden impact on research results. Furthermore, by comprehensive genomic profiling of 8 most common used urothelial cell lines and comparing them to previous publications, we found that regardless of similar short tandem repeat (STR) profile, driver gene loss in cell lines could happen by random mutation. Our results suggest that clinical research using urothelial carcinoma cell lines might be influenced by cell line genotypes which could only be determined by next-generation sequencing. Meanwhile, this study indicates that the conditionally reprogrammed cells (CRCs), which closely resemble original tumor tissue, might represent a better alternative for in vitro research, which may be better used for personalized medicine.

Endometriotic cell culture contamination and authenticity: a source of bias in in vitro research?

STUDY QUESTION Are the primary cell cultures and cell lines used in endometriosis research of sufficient quality? SUMMARY ANSWER Primary cells used in endometriosis research lack purity and phenotypic characterisation, and cell lines are not genotypically authenticated. WHAT IS KNOWN ALREADY The poor reproducibility of in vitro research and the lack of authenticity of the cell lines used represent reasons of concern in the field of reproductive biology and endometriosis research. STUDY DESIGN, SIZE, DURATION In the present study, past in vitro research in the field of endometriosis was systematically reviewed to determine whether the appropriate quality controls were considered. In addition, we explored the performance of Paired Box 2 (Pax2) as an endometrium specific marker in endometrial and endometriotic primary cell cultures; we also characterised the most diffused endometriosis cell lines with respect to important markers including the short tandem repeat (STR) profile. PARTICIPANTS/MATERIALS, SETTING, METHODS Literature review part: almost 300 published protocols describing the isolation and creation of primary cell cultures from endometriosis were reviewed. Wet-lab part: primary cells isolated from 13 endometriosis patients were analysed by immunohistochemistry, immunofluorescence and FACS for the expression of Pax2. Cell lines Z11 and Z12, the most diffused endometriosis cell lines, were characterised with respect to the expression of Pax2, steroid hormone receptors and STR profile. MAIN RESULTS AND THE ROLE OF CHANCE From the literature review work, we underscored the lack of sufficient cell purity and phenotypic characterisation of primary cell cultures, which present high risk of contaminations from surrounding non-endometriotic tissues. Past work based on the use of cell lines was reviewed as well, and it emerged that cell line authentication was never performed. In an effort to address these weaknesses for future research, we present data on the performance of Pax2, a suitable marker to exclude ovarian (and other non-endometrial) cell contaminations from primary cell cultures; STR profiles of cell lines Z11 and Z12 were analysed and indicated that the cells were authentic. These profiles are now available for authentication purposes to researchers wishing to perform experiments with these cells. A quality control pipeline to assure sufficient quality of in vitro research in the field of reproductive biology and endometriosis is proposed. We encourage scientists, research institutes, journal reviewers, editors and funding bodies to raise awareness of the problem and adopt appropriate policies to solve it in the future. LARGE-SCALE DATA STR profiles of cell lines Z11 and Z12 are deposited at the Cellosaurus database—web.expasy.org. LIMITATIONS, REASONS FOR CAUTION There may be additional markers suitable to assess cell quality. WIDER IMPLICATIONS OF THE FINDINGS Future in vitro research in endometriosis and the reliability of outcomes can be improved by using the recommendations presented in this study. STUDY FUNDING/COMPETING INTEREST(S) The study was partly financed by the ‘Stichting Fertility Foundation’ (The Netherlands). The authors declare no existing conflict of interest. TRIAL REGISTRATION NUMBER Non-applicable.

Single Cell Analysis of Circulating Endothelial Cells in Allogeneic Hematopoietic Stem Cell Transplant; To Whom Do They Belong: Host or Donor?

Background. We recently reported that Circulating Endothelial Cell (CEC) count changes represent a promising marker to monitor endothelial damage in patients undergoing allogeneic hematopoietic stem cell transplant (allo-HSCT), potentially becoming a valuable tool in the diagnostic definition of GVHD. Besides confirming an increase of CEC counts at GVHD onset, we repeatedly documented at time of engraftment statistically significant higher numbers of CEC in patients who will not manifest GVHD in comparison to patients in which GVHD will be diagnosed (Transplantation 2014,98:706-12; Bone Marrow Transplantation 2017,52:1637-42; Scientific Reports 2019,9:1-12). Recent knowledges in organ transplant pointed out that endothelial cells from the grafted organ, besides being a continuous source of alloantigens, can downregulate alloreactivity exerting tolerogenic responses. By inference to the allo-HSCT field, it could be envisaged that presence of donor CEC could induce protective effects on alloreactivity. Methods. We planned a study to test the hypothesis that at time of engraftment, CEC present in peripheral blood (PB), besides coming from cells shedding from patient vasculature, could partly belong to donor, originating from the cellular graft. Therefore, in an exploratory set, we performed FISH analysis on flowcytometry-sorted CEC (CD45neg/CD34bright/CD146pos, Lyotube #623920, BD Biosciences) (n=3) and on whole PB derived culture-expanded CEC (n=3) (EGM-2 BulletKit, Lonza), obtained at engraftment in sex-mismatched allo-HSCT. In the confirmatory set (n=15), single CEC were recovered from PB, at engraftment (T1) and at 90 days (T2) after allo-HSCT, through the DEPArrayTM technology (Menarini Silicon Biosystems), after preliminary bulk separation step carried out with the CellSearch® System. Single recovered CEC was whole genome amplified (Ampli1™ WGA Kit) and short tandem repeat (STR) profile determined (Ampli 1TM STR kit) on each single CEC. To confirm host/donor origin, single CEC STR profile was compared to that determined on patient and donor cells before allo-HSCT. Moreover, donor CEC presence was evaluated by CISH analysis on formaline fixed and paraffin-embedded biopsy sections obtained at least three months after sex mismatched allo-HSCT. Results. By positive findings of the exploratory set, we proved, at the single cell level in the confirmatory set, the presence of donor CEC at engraftment (T1) in 4 out of 15 patients (Table 1). Of them, 2 did not manifested GVHD, despite a GVHD risk score of 2, and the other 2 presented GVHD grade I. On the contrary, among the 10 patients in whom no donor CEC were detected, 6 experienced GVHD grade II-III, while 4 did not manifested GVHD, despite a 1-3 GVHD risk score. Conclusions. Our data represent the proof of principle that donor CEC may flow in host PB early on from hematopoietic recovery and seldom persist thereafter at steady-state conditions, being potentially embedded in host vascular wall. These puzzling findings suggest that neovascularization takes place in parallel with hematopoietic engraftment and could provide further clues on shedding light on tissue tolerance in the context of GVHD, opening up paradoxical scenarios on the protective role potentially played by donor CEC. Disclosures Fontana: Menarini Silicon Biosystem: Employment. Rotta:BD Biosciences Italia: Employment. Manaresi:Menarini SIlicon Biosystem: Employment, Membership on an entity's Board of Directors or advisory committees.

DNA Testing Reveals the Putative Identity of JB55, a 19th Century Vampire Buried in Griswold, Connecticut

In 1990 in Griswold, Connecticut, archaeologists excavated a burial found in a “skull and crossbones” orientation. The lid of the 19th century coffin had brass tacks that spelled “JB55”, the initials of the person lying there and age at death. JB55 had evidence of chronic pulmonary infection, perhaps tuberculosis. It is possible that JB55 was deemed a vampire due to his disease, and therefore had to be “killed” by mutilating his corpse. In an attempt to reveal the identity of JB55, DNA testing was performed. Ancestry informative single nucleotide polymorphism (SNP) analysis using the Precision ID Ancestry Panel indicated European ancestry. A full Y-chromosomal short tandem repeat (Y-STR) profile was obtained, belonging to haplogroup R1b. When the Y-STR profile was searched in the publicly accessible FamilyTreeDNA R1b Project website, the two closest matches had the surname “Barber”. A search of historical records led to a death notice mentioning John Barber, whose son Nathan Barber was buried in Griswold in 1826. The description of Nathan Barber closely fits the burial of “NB13,” found near JB55. By applying modern forensic DNA tools to a historical mystery, the identity of JB55 as John Barber, the 19th century Connecticut vampire, has been revealed.