Cass Nordmann - Dr. Sandra Demaria - Week 6

 I wasn’t able to work on my own project much this week because I’m unfortunately still waiting on the collagen I ordered. It’s not coming until the 18^th so I won’t have a lot of time to gather data, but hopefully, I’ll have at least some indication of how the results are looking by the end of my time here. I’ve been asking around and seeing if I can get collagen from another lab here so maybe I can at least get some later today or early next week and have a few more days to grow my organoids. While I wait, I’ve been continuing to research the literature to decide on some aspects of my experiment and see what results any similar work has yielded. I found one paper with a similar experiment to mine in which cells grown in Matrigel formed much more compact organoids than those grown in collagen, which tended to migrate out. However, as far as I can tell, the authors of this paper didn’t use anything to inhibit the RhoA/ROCK pathway involved in actin contraction. RhoA/ROCK inhibitors are often included in organoid media to improve organoid formation, which could make a difference when I try to grow organoids in collagen. I’ve done most of the prep I need to do before any lab work including calculating volumes of solutions needed and adapting the Demaria lab’s protocol for organoid passaging to use collagen rather than a basement membrane extract.

On Monday I was able to go back to the histology core with the medical students and the new postdoc, Xiao. I got to participate in the training for H&E staining, which was surprisingly simple. The process is essentially just moving a caddy with your slides from one tub of liquid to another and then waiting for a given timespan or dipping it in a certain number of times. The only part of the process that was at all challenging was applying the coverslip to the slide after the staining is complete, but even that was fairly easy and just requires patience, so you don’t introduce bubbles into the fixative as you lower the slip onto the slide. I’ve never done any histology or even understood how to interpret histology images before coming here for the summer, so this has been a really valuable takeaway for me. I would be interested in doing some more histology on the spheroids my lab has back in Ithaca. Ithaca doesn’t have a histology core like the one at Weill, so it’ll be more challenging without everything set up for me, but I think it could be really informative about how closely our spheroids model true tumor structure.

Later that same day, Marvin showed me another sample he was preparing for histology. Normally, tissue samples being sent off for histology are placed in plastic cassettes where they are fixed in paraffin wax before being cut and stained. The cassettes have gaps to allow the solutions used during paraffinization to penetrate the sample. For smaller samples, rather than gaps, the cassettes have a fine mesh over the openings. However, organoids are so small that even the mesh cannot contain them. Instead, before histology, organoids are mixed in a solution of thrombin and fibrinogen, which react to form a “clot” that encases the sample of interest. The entire clot is then placed in the cassette for paraffinization. I wasn’t able to attend the paraffinization training that occurred later on Monday and Tuesday due to space limitations, but I did at least get to see this part of the process.

On Wednesday, some slides that Diego and Marvin had sent off to another core facility for slicing and staining came back so I was able to view the slides with them. The microscope in the cell in the biohood room here isn’t the best quality so later in the day when Dr. Demaria was available she took us all to the pathology lab to look at the slides with her. The slides were of Dignity organoids, which is the line which Diego was having trouble getting RNA from and he wanted to see if high confluency was leading to necrosis in these samples. Interestingly, Dr. Demaria said that she didn’t see any necrosis in the slides that we were looking at but that she saw a phenotype that she called “signet ring cells.” These cells overproduce mucin in their cytoplasm which creates a large vacuole in the cell that takes up much of its volume and compresses the nucleus, forming a ring in the middle of the cell with the rest of the features squished on the edges. It’s a relatively rare occurrence in breast cancer and not something that anybody expected so further histology will probably be done to confirm that it is in fact mucin.