Presenting a short talk and poster on Intestinal Stem Cells and Colon Cancer: Biology to Therapy conference

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Poster for the AACR conference. Trajectory analysis of mouse large intestine.

Trajectory space analysis: Leveraging computational models and single cell RNAseq to understand genetic programs defining intestinal lineages and infer colon stem cells in mouse

High-dimensional single cell profiling holds the potential to elucidate developmental sequences and define genetic programs directing cell lineages as well as determine putative progenitor cell types.

Existing algorithms have limited ability to elucidate branching developmental paths or to identify multiple branch points in an unsupervised manner. We introduce the concept of “trajectory space”, in which cells are defined not by their phenotype but by their distance along nearest neighbor trajectories to every other cell in a population. We implement “trajectory space” in a tSpace algorithm, and show that multidimensional profiling of small intestine cells in trajectory space, with minimal user input, allows unsupervised reconstruction of developmental sequences from intestinal stem cells to specialized epithelial phenotypes. tSpace delineates absorptive/enterocyte and secretory/enteroendocrine (EE) development, both arising from Lgr5⁺ crypt base columnar (CBC) cells, and positions cell types in developmentally meaningful relations. tSpace clearly positions short lived EE Dll1-expressing progenitors in trajectory space between CBC cells and mature EE populations, outperforming existing analytical tools (t-SNE, SPADE), which failed to define these cells either as a discrete subset or as a precursor population. Furthermore, our analysis reveals patterns of gene expression mirroring observations from decades of research on intestinal development.

Utilizing power of tSpace we identify three transcription modules that specify cell fate within CBC progenitors. The module consisting of Foxa2, Foxa3, Neurog3, Sox4, Sox9 is expressed by early cells but maintained in late CBC cells selectively in the EE branch; these transcription factors have been associated with endocrine and pancreatic development and may coordinate secretory pathways within intestinal enteroendocrine cells. Two modules are expressed preferentially in the enterocyte branch. One activates lipid and cholesterol metabolism, known to be important for mature enterocytes, while other is associated with Nfe2l2/Nrf2-antioxidant response element (ARE) pathway. The trajectory analysis shows that rapidly proliferating subset of CBC and transit amplifying cells are already heterogeneous and express gene programs leading to secretory vs. absorptive phenotypes.

Furthermore, we apply similar approaches to understand developmental sequences within mouse colon crypt.

We believe that tSpace will prove useful to the rapidly growing field of singe cell analysis and that our intestinal analysis can be a resource for further studies of intestinal development. More can be seen in pre-print here.

Single Cell Omics, Stockholm May 26th

Branching developmental pathways through high dimensional single cell analysis in trajectory space: application to the tumor environment and tumor control of the immune response

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Cancer immunotherapy has proven successful with new generations of drugs (e.g. checkpoint inhibitors) that target lymphocytes and their surface molecules, however only small fraction of the patients benefits from these drugs. Thus, it is important to uncover additional mechanisms of tumor immune evasion. Lymphocytes are recruited into tumor by endothelial cells (ECs), which are the interface between the tumor and circulating blood. Tumors induce specialized ECs, known as high endothelial venules (HEV). Induction of HEVs in tumors correlates with favorable prognosis, but many tumors use HEVs to recruit immune suppressive cells. Tumor microenvironment through conditioning of the local endothelium trafficking programs mediates recruitment of immune suppressive lymphocytes and enhances carcinogenesis. Interaction of tumors may also alter the imprinting of lymphocyte adhesion/trafficking programs in tumor draining lymph nodes (LN) either through effects on tumor derived dendritic cells or directly when the LN is invaded by metastatic cells. We take advantage of mass cytometry (CyTOF) and single cell RNA-Seq to monitor the induction and regulation of EC and leukocyte trafficking receptors in B16, B16 OVA (more immunogenic), 4T1 and colon mouse tumors. High dimensional data, rich in information, is analyzed using an innovative single cell analysis algorithm, which aligns cells in developmental trajectories using protein or gene expression(s) of single cells. This algorithm has the potential to reveal, for the first time in an unsupervised manner, complex branching and allows the detection of new functionally important cells or specific transient cell populations. #Ksomics

New publication in Cancer Research: Western diet deregulates bile acid homeostasis, cell proliferation and tumorigenesis in colon.

Western-style diets (WD) high in fat and scarce in fiber and vitamin D increase risks of colorectal cancer (CRC). Here we performed a long-term diet study in mice to follow tumorigenesis and characterize structural and metabolic changes in colon mucosa associated with WD and predisposition to CRC. WD increased colon tumor numbers and mucosa proteomic analysis indicated severe deregulation of intracellular bile acid (BA) homeostasis and activation of cell proliferation. WD also increased crypt depth and colon cell proliferation. Despite increased luminal BA, colonocytes from WD-fed mice exhibited decreased expression of the BA transporters FABP6, OSTβ and ASBT and decreased concentrations of secondary BA deoxycholic acid and lithocholic acid, indicating reduced activity of the nuclear BA receptor FXR. Overall, our results suggest that WD increases cancer risk by FXR inactivation leading to BA deregulation and increased colon cell proliferation.

https://www.ncbi.nlm.nih.gov/pubmed/28416481

Mutator phenotype versus growth advantage in cancer

I’ve been fascinated by DNA and genetics since reading Jurassic park as a kid. Later, when I started with studies I couldn't wait until I was ready to engage in actual course on genetics. At that time my attention was drawn to DNA mismatch repair (MMR). A highly conserved mechanism, which exists in every alive cell and its primary role is to detect mismatches in DNA that will become mutations if not corrected prior cell divides.

Mismatch repair mechanism consist of rather simple steps. First, MSH2-MSH6 detect mismatch. Second, MLH1-PMS2 is engaged and that signals EXOI to cut out mismatch. New gap in DNA strand is re-sythetized by DNA polymerase.

Mismatch repair mechanism consist of rather simple steps. First, MSH2-MSH6 detects mismatch. Second, MLH1-PMS2 is engaged and that signals EXOI to cut out mismatch. New gap in DNA strand is re-sythetized by DNA polymerase.

Taken high conservation from bacteria to humans, somebody would say if you mess up with MMR life would not be possible, but… none of MMR genes when inactivated are lethal, nevertheless inactivation of even just one of two gene copies will speed up cancer development. A syndrome named after Henry Lynch is associated with inherited mutations in MMR genes. Individuals with inherited mutations in MMR genes have predisposition to develop mainly gastrointestinal cancers and women endometrial cancer.

Why is that? Well, many would say isn’t it obvious? Deficient mismatch repair leads to increased mutation rate which at the end causes cancer. I encounter this answer again and again. But increased mutability isn’t necessarily a bad thing. Without mutations life wouldn’t evolve. So why is it then that MMR deficiency is a shortcut to cancer? A less known function of MMR, and quite often shadowed by mutator phenotype, is its role in signalling apoptosis or cell death.

Healthy cells when faced with irreparable mismatch, and DNA damage in general, will either enter senescence (something like retirement, cell is still alive but not dividing anymore), or undergo apoptosis - death. MMR deficient cells, will gather more mismatches because of obvious reasons, but these cells will not undergo senescence neither trigger apoptosis. They will divide and mismatches will become mutations, of which some mutation combinations will be lethal for cell, some will live. The latter is just matter of statistics and combinatorics which combination will evolve into cancer.

In other words, inactive MMR is a shortcut to cancer because MMR deficient cells have selective growth advantage over healthy cells, and mutator phenotype is just speeding variability of cells.

Most chemotherapeutic drugs work on principle to induce apoptosis in cancer cells, but that strategy does not work in MMR deficient cancer cells, because they are unable to signal apoptosis. At present moment only gene therapy, which would restore MMR activity, accompanied by cytostatic would be efficient approach. In light of not-that-successful experiments and quite high risks of gene therapy, my opinion is that understanding mechanisms by which MMR signals apoptosis in greater detail would give a clue towards new drugging aproaches for MMR deficient cancers.

Amazing Cancer Race 2015 - Provocative questions pit-stop

When sun starts to break heavy gray clouds in Finland it's time for Amazing Cancer Race - a finale of a problem based learning course CancerBio Summer School. I participated in the course five years ago, and since then organized it four consecutive years. Originally it was Juha's course, but Maral, my colleague and me every year added a twist and with Juha's support course grew in popularity.

Shortly, course revolves around hallmarks of cancer, proposed by Bob Weinberg & Doug Hanahan. Up to 20 students can participate and they are split in pairs. Each pair have to approach one hallmark, define problems in that field and open discussion with the rest of the group. On each session an expert in the field is invited to maintain levels of discussion and fill in scientific facts if needed.

During the course each session is graded by the students and point collected on your activity and presentation are used later for your line up in Amazing Cancer Race (ACR), which is a grad finale of the course.

We developed and tuned over past five years Amazing Cancer Race. Idea is to incorporate science, healthy activity and creativity into one competition. Students run from one pit-stop to another, and location of each pit-stop is reveled after decoding a riddle-map. Once on a pit-stop you can play a game based on cards developed by us, which are linked to hallmarks of cancer and override difficulties that were placed in front of you by previous teams, or you can block team that arrives after you, or you can buy time... In addition to that on each pit-stop you have two types of games to play: one requires brain activity another is more physical - you can choose, and on some pit-stops you have to actually do something special.

Some teams were creative and made costumes for the race...

This year Juha and I were on a pit-stop that was named Provocative questions. National Cancer Institute (NCI) has a list of provocative questions (PQ), current 12 are here. Aim of these questions is to address and tackle some specific topics difficult to resolve, or topic that would need additional attention. In times like these, when scientific funding is scarce and difficult to get we thought that this year on our pit-stop students have to choose one question among these 12 and propose more specific aim of their research, suggest methodology and last but not least, highlight impact of their research. All that in 20 minutes... Tough and challenging, but they had prior opportunity to read PQs.

It was really interesting to participate in discussions and see how many times beautiful ideas can be born out of pressure.

... inspired by their research. NHL or Non-Hodgkin lymphoma? 

The most frequently chosen question was about studying properties of cells from a pre-malignant field, usually surrounding cancers, in order to develop strategies to inhibit the development of future cancer. One of the answers was different from others; it combined biology and mathematical modeling. I loved the idea of developing a model based on biological data. Their concept was not polished or did not go deep into biology, but still it was a refreshing view.

Second question that was picked commonly investigated mechanisms of action if standard-of-care classic therapies would complement immunotherapy. It is interesting to note how students aimed for questions that are not heavily researched here in Helsinki.

One team choose to tackle cancers, which can be dormant or indolent for long periods. This topic is extremely interesting because it is unknown which mechanisms maintain these stable states. Their approach involved developing animal model for studying progression of cancer and dissection of cancer surrounding/neighboring tissues, which may influence these long dormant periods. I would add that this topic if approached with cutting-edge single cell -omics techniques and combined with computer modeling could soon provide some answers.

A special guest this year was Dr.Laura Soucek. She was on another pit-stop judging students when they had to pitch their project either to a scientific committee or to two rich ladies. Many teams chose two rich ladies, not thinking that rich ladies may be more difficult to persuade than scientific committee. Some biological questions can be a challenge to explain in a more general way. This was extremely valuable eye opener to me; we all are with our heads many times deep into science forgetting that science should reach wide audience, and not be exclusive for academics. All tax payers have right to be informed and involved in most recent scientific findings.