 Yeah, good afternoon everybody The almost I have to say thank you for organizing this really nice symposium necessary for reproducibility of data and It's a really good platform to get to give also a good view of SLI repository Where I work since two decades already And but it is my my last year so I go in retirement led next year. It's a nice Opportunity to meet all you nice people from my clock and from the standard Developing organization and so a real pleasure for me Okay, and fascinating We did not talk to each other But it is perfect fitting of our talks together because I have a really a minor problem obviously minor problem to tell you today But it's the consequences are really severe and I will show in a few minutes, so Yeah, finally cancer cell lines Very nice introduction to cell lines and the cross-contamination situation cell lines have been so this is really a beautiful tool to work on cancer research because because they have been the backbone on cancer research for for for decades and So finally I give also a very very short introduction into the situation So this is a picture of mindful for these nice work of Nicole Surin and What you see is We can We need reproducible scientific data because it is shown you waste too much money It's nearly one billion US dollar a year. It's wasted on wrong cell lines Seline models and even these papers going out of this scenarios and they are missing and this is What we have to to act on and what you see here is Where this patient you get the tumor side biopsy You can establish a cell line from as long in this green area You use authenticated cell lines or even sub clones of this main Population you will get the valid data So the opposite is nicely explained by Amanda the cross-contamination situation invading of a foreign cell into the establishment situation, so this is cross-contamination at source and If there is a little bit proliferation Advantage of the foreign cells, so it will be overgrown. It's just a question of time So finally interchange and also mislabeling also contribute To this situation, but what is really connected and this is also endangerment for reproducible scientific data is this section I want to focus now on and the end end endangerment is done because or by chromosomal instability I will give an example of this and also by micro satellite instability So these are the main drivers of the genetic drift A lot of science scientists call this genetic drift evolution in vitro So but this is not no real evolution because it's not connected to any natural selection Processes this is really genetic drifting in vitro conditions okay, and Yeah, finally, I will go further now to to explain What loss of heterozoagosity in the first line is meaning to a cell because when we do str. Typing's When we are lucky we get the reference profile from a completely heterozygous cell and So carrying maternal and paternal chromosomes. There is a nice de pluit or heterozygous str profile and Yes, somehow there are different loads of Causing events of Copy loss loss of heterozoagosity or copy neutral. This is just meaning that the whole Maternal or paternal chromosome is lost in it to avoid the my top by to Metotic catastrophe The resting one is only doubled, but this is finally going to to convert the double sickness into a single str Profile and I will not go into deep today in In the micro satellite instability situation because micro satellite instability is caused by the deficiency of the mmr system DNA repair system and Which is normally wiping out all our replication errors during DNA replication and This is this drifting can cause Third and or multi alleles at what and this is not meaning that this is occurring in a single cell even if cancer cells are tree or tetraploid and These are sight lines all the sight lines Can carry 10 and 11 or 10 12 or 11 and 12? So these are the situations, but this is not present in a single cell We have done a lot of single cell Authentications and we know that you normally get only a deployed situation Or profile from from these cells okay, and this is always connected to the events or to the phenomena of Loss of heterozoagosity as well as micro satellite instability because there is Yeah, silent danger by wrong cultivation Techniques and this is an image of Karzai et al. So he's working at the right-hand cell bank and I Forgot the citation sorry for that and so he Experienced that if he is growing a culture to complete density Proliferation is stopped and has to start again in the next Passaging so and if you have done this too often if you have an overpassage cell line Then you will work out. So the the sight lines within the population which which are always present They get a chance to fit better the new conditions and then you have the outgrowth of a specific Single clone or a sideline of of cell and and that means if you have an outgrowth you have lost Lot of properties from the whole cell population and this is a problem and Now I will go to in an example because memories are nice examples lasting longer and So there's a pro from from the lab of Perot There's a report that the cell line thp1 Which is an acute myeloid leukemia cell line And it's characterized by chromosomal translocation and it is fusion and two different parts of a transcription factor together and this is and they is doing in Reprogramming in vivo and this is the cause for leukemia in And finally so this is one of the most important cell lines for studying Yeah, leukemia and which genes are affected by the transfusion gene and So they worked Noronia Nandita no, no, they obtained Thp1 from two major bioprositories the SDR profiles who were really very very close together and But finally they found out that the discrepancies they worked out Mainly were due to the heterozygosity and finally They argued that one of the thp1 cell lines is not any more reliable modi for studying leukemia And this although Authentication has taken place so this is a real problem and now I will Try to convince you what they have done so they did from The thp1 cell line from repository a and b they did whole genome sequencing and Finally, they could work out by a new tool, which is electronic SNP choreotyping They worked out for example that chromosome six at least in part is completely Homozygous which is indicated in in a single blue color and while The cell line from repository B is heterozygous for this area and now that you are really drastic Situation is found on chromosome 13. So you see that from repository a The whole chromosome or the long arm of chromosome 13 at least is homozygous while from Repository B this part of the chromosome 13 is completely heterozygous and that means so Authentication has taken place. They are from major cell repositories all claim These cell lines are authentic and useful for our research and finally so I skipped just this in this image and When they worked out with the transchrome transcriptome NGS data they can they had Generated a heat map of this of AML related genes. So that is mean the transfusion or the fuse transcription factor is affecting a lot of other genes and look at this so The row on the right side from the repository B. These are all the target genes which are Upregulated by that by the chromosomal translocation and this is not found anymore in the cell line in THP one from Repository A Okay, and this is Yeah Finally a little bit disturbing the picture Amanda and Jamie has drawn this morning So it's not always black and white and this is completely disturbing a little bit But there are ways out because these findings has also initiated to find and to build up new tools at our repository and first of all The first step should be have a look into Salazar's because and I most respect to your work Tracing all this str profiles to single papers and this is an amazing work. I have really Much respect to do himself and here is already shown that there are the chromosome 13 and 13 here and comes on six here and That there are the differences. So the loss of heterozygosity is indicated and at a lot Yeah, a lot of other salary repositories and the sad story in this case only is That only three repositories are dealing with the right Yeah type of THP one While 12 others are not and that means that 90 percent of I guess So this is an estimation that 90 percent of all THP one cell lines out from major Repositories are not suitable to do leukemia research with regard to acute myelot leukemia Okay, and to to give further. Yeah, this is our own Study, so if you use different algorithms to work out these similarity matches between different cell lines and You work out that only tenable algorithm is giving the difference and not the this one of John Masters But okay anyway There is a we thought we have to to come in into action and what we did is finally we created Yeah, I have to also mention Laura Stainpass. She is new head of our departments in three years and One of the first things we have done then is was creating cell dive and cell dive means that we have meanwhile 160 cell lines are Yeah read out for the transcriptome by NGS techniques and So we have even of course we have our own SDR profile search, which is also public So anybody can use it and you see I just have listed here all the different THP one Cell lines including so in red these are the LOH Problems in in cell lines if they are there and For a much better control because we have the differences these are old cell lines They have been established in the 90s and genetic drift is taking place. So this is biology finally and so We we put on the transcriptome data on our hub and what you can do now I bring you really Quickly through you can give in for example, you can choose and in tighter T So so far we have have been focused on leukemia and lymphoma cell lines, but we have also Meanwhile breast cancer cell line panel sequenced and this is will be updated in this year and so so finally you can Give in the genes you are interested and you can follow up in all cell lines distributed exclusively by DSMZ you see here THP one is Completely positive for one of the leukemia genes DNMT-3B, MyS3, HoxA7 and so on so this is helping The customers that because they can't look Into the physiology of the cell and can check out If this is the right system or not and if the genes expressed I'm interested Okay, finally, I have to speed up a little bit There yeah, cell lines have been the backbone of science or cancer research of the last century this century Because we have much better knowledge about how a solid tumor for example is looking like we have so much Bicep in the cells we have a scaffold and the vessels and in all the this stuff and this This These cells that are contributing because there's cross-talking between the tumor cells in blue this is a hypoxic center, which is dying off normally and loads of immune cells and these cancer associated fibroblasts of calf cells Okay, and I was really impressed by Work from the Netherlands Kerstedal they published in cell genomics and this is really an amazing work because What they did is they isolated primary Colorectal cells Using CRISPR-Cas9 technology to set up the most found mutations in these cells So this is complete knockout of P53 and Specific cells and they so you see the different mutations they put Single or twice or triple into the cells and and they are marked by by colors here And they put also this is a retroviral Induced barcode for identification of the cells so and then they let these cells grow and They formed nice organoids and then finally they did whole genome sequencing in multiple time points during evolution evolution is meaning to grow from single cells to this Organoids and Finally, they what they did then is using the VGS data They have been able to work out copy number variation off of these all the single cells a huge work and single single nucleotide variants Identification so real mutations in the genome Frameshift stops and and so on and finally tracing and All these developments in one dendrogram and this is looking like this and the fascinating point is coming now because these markers chromosome for for colorectal colorectal cancer loss of chromosome 18 and also later on chromosome Part of course home for both of these are lost then they give rise to the most aggressive Colorectal cancer you can get so really aggressive CRC's and So this was a really convincing work They they did because they really mimic Tumor evolution in vitro. So this is a very new step and this is also Yeah motivating us at the SMZ to also look for a new Can some models, but first of all I have to to mention the main players so finally Jesse Burnham and also taught go up from from the Broad Institute and find financial is supported with very well. So this is 2015 and finally and they formed a community of Yeah, high-level labs. I should say and they Worked in the human cancer model model initiative and And these models are explicitly Distributed by ATC by no other by resources But so finally William Sellers CEO of Novartis before and he is now the scientific director of the Cancer cell line factory within the Broad Institute in the right hand of him Monit Singh They have the aim to converse any tumour type into a cell lines for read or Organized model and they are doing this by Genomic profiling of donated cancer cancer tissue and this not after 50 passages This is done after passage two three or four and so they do then screening a dozen dozens of cell culture condition combinations huge work and So finally to to grow the cells and in case of successful cultivation, they do a whole transcriptome sequencing again and Then they do a comparison of the original tumour with the clinical data of the patient and They go then for the next neighbor and this is enabling a very very close model to the original tumour and finally There's a selection of the best cultural condition based on tumour Genomics Finally, they are really successful. I never expected that they as Work out so a tremendous amount of Cell lines from rare and really difficult Tumors and they so over 700 models they got so far and Look at this 47 so nearly half of all these 700 models are of 3d and organoid Morphology and so this is showing us that this will be part of the future also at DSM Z so we have a nice cooperation and I will show that we have due to this collaboration last year. We have been able to complete accession five new cell lines and you see this is New for eight. Yes, it's a long name, but we clearly want to Indicate this is CCLF models so one of the new ones and Yeah, you see this is growing at its face or little organoids are always present starting from from finally single cells but as a biorepository we are dealing with Cell lines which should be infinite because otherwise it's so much work to get them accessioned into cell bank It's really expensive to do so and finally if they are on a primary state They will die off after reaching a flick limitation and therefore I have seen the need to establish a New version of the so-called trap protocol. This is measuring telomerase activity in in in in cells in or any mass vertebrate cells I have to say and Because there is a problem the primary cells There is an end replication problem at the telomeres and every in every cell division They lose this single strand part and this up to 70 80 90 divisions is needed and then the telomere is Gone for example, and then the whole chromosome gets to be in stable and this is Leading the cells into a crisis and then they finally die off okay, and so and we would discuss this with CCLF already and they promised out that they have been really Very successful in to induce telomerase activity by the culture conditions. I Have to be honest. I did not believe this in front of the project and so I finally with my beautiful technician sick family and We adopted an original protocol from the 90s into a fluorescent version of the trap essay by doing a lot of new things and We have been able to create a very very sensitive Essay, which is this is the analyzes in the capillary capillary electrophoresis and you can see nicely every the telomere length so this is the telomere repeats GGG ATT and We can finally work out How active is cell line or the telomeres activity isn't Within a cell line, okay, and Finally So these are controls positive control negative control. This is a cell line, which is there's another way to to prolong The telomeres by homologous homologous Recombination, but this is control to another cell line, which is not and this is heat and inactivation And you see from two of the five CCLF cell lines, which are all positive for telomerase activity That with that they differ a little bit in the condition, but we get the nicest free size only using nine thousand cells in this essay which will be Published during this year in biotechnics that we can go down to to almost 50 cells or maybe less and To get clear signals of telomerase activity Finally, I come to my to to the end So we are happy that we have the first new cancer cell line models in our collection and Yeah, this is a short summary at the end So we need new qualities for increased reliability of scientific data I have shown overgrown overpassaged old cell lines. They can have a problem and and So finally SCR 70 as a reference technique is indispensable There's an endangerment by ex vivo in vitro LOH and MSI There is a need for evaluation I have to convince almost a little bit to work together on this topic to get the most Reliable as they have reference profiles we which should show maximum of heterozygosity and I See the need of e-snip carry-out typing for very exclusive models to follow up copy number variations and This address also to dear young all the new models are free of FPS and They have an improved biological Relevance because of the the the neighborhood they're very close together and so basic research on on tumour cell development should be better and Also drug development should be meet better targets doing this with a new new tumour models. So finally We are going to at the SMZ to going to extension Our cell dive data to to further tumour entities Next will be colorectal cancer Then we have pros promising media matey mediated reprogramming and organoid differentiation technology Implemented by by Laura stand pass and Expanding we are going to expand the CCLF collection of difficult and rare tumour models Okay, this is my former team my my PhDs. So at the end of my Yeah, working time My my technicians and I have to mention of course Laura stand pass with a lot of new input in our department Sonja Abert Abert Responsible for the CCLF collection and Claudia Pomarenke And she is our be bio bioinformatic expert and caught up off which is Yeah, doing all the risk assessments we need for for new cell lines to look for viruses other infections Okay, thank you so much for listening and Let's see Hi, I'm a bit of an off-the-wall question Where do you see automation in these new sorts of models use use of automation to actually Standardize and get the reproducibility going Yeah, not different not not easy to answer finally I see Automatically on all high put through techniques, which means sequencing and all this stuff so there's but Finally, they're still Good manual work Affordable and necessary for from from our technicians because Cultivation protocols even for these a little bit difficult to culture. This is indicated in our catalog that the CCLF models are not easy but we give clear guidelines how to handle them and This will be work. You never I'm pretty sure this can can never be automated Thank you for your presentation. I'm very grateful. You mentioned one word Karyotyping I'm a geneticist by training so I Have the feeling very few people do that still today though It's one of the oldest methods in cell culture to realize what's going on. You know, I remember a paper for 1948 or something Telling us whoever uses immortalized cells that Cells are changing and you can see it on the career type Now we have these modern methods with next-generation sequencing and other people think oh that gives is a solution But I don't agree They what you get in a NGS is an average You get the dominant whatever in your population is heterogeneic. You get a sequence. Yes But it does not represent The cell culture which in front which is in front of you I have to correct this a little bit because I'm a great fan of Karyotyping and I miss my colleague Rod McLeod. He was a really nice person from Scotland and he did all these Karyotyping in our lab in at a very high level including the sky Staining of this different karyotypes Finally the end NGS technology has developed meanwhile so far that we can really nicely and more precisely than ever cover the reads to the density of Yeah, estimate template which is and this is the basics for e-karyotyping or snip karyotyping for the estimation of copy number Variations in in cell lines. So this is really and finally There is no other way because all the cytogenesis are gone So there's nobody who is Really has a good education in this technology anymore I'm sad about this. So I'm a big fan of karyotyping. Yes