Best of the Best Gen Surg - Development of postoperative local tumors and distant metastases in diet, genetics and microbiome-dependent in a mouse model of colorectal cancer recurrence - Dr. Morgan

All right, we're gonna go on now to the SSAT session. Uh, the SSAT, the first paper is presented by Doctor Ryan Morgan. And that paper is entitled Development of Postoperative Local Tumors and Distant Metastasis in Diet, Genetics, and microbiome Deependent in a mouse model of colorectal cancer recurrence. So we'll have the uh. A video by Doctor Morgan. Thank you for the opportunity to present. My name is Ryan Morgan. I'm one of the uh general surgery residents from the University of Chicago, and I'll be talking about our work from the Shogun Lab. Uh, colorectal cancer is a leading cause of cancer deaths, particularly in cases of recurrence, and traditional paradigms have suggested that recurrences are due to either incomplete resection or occult metastases left behind at the time of surgery. Multiple studies have raised the possibility, however, that exfoliated cancer cells in the lumen of the bowel may be a source of recurrence as well. In the 80s, UMLB showed that these cells are present and viable at the time of surgery. A subsequent study found that these experimental cells could cross a watertight anastomosis in a mouse model. Uh, further, the role of these cells is supported by clinical data that shows that a rectal washout decreases rates of local recurrence after rectal cancer resection. And while these studies don't necessarily show, don't necessarily, uh, explain the exact mechanism underlying this, uh, phenomenon, they suggest that these cells may be a source of recurrence. Uh, these data, along with others, have led us to develop a novel model of recurrence. When the primary colorectal tumor is resected, these exfoliated cells remain behind within the lumen of the bowel, and they're able to interact with the anastomotic microenvironment. Multiple factors can influence this environment, including diet, the microbiome, and the tumor genetic background itself. We propose that the interaction of these factors in the healing anastomosis promotes the migration of these cells to cause both local and distant recurrences. Uh, specific to the study, our hypothesis is that a high fat diet and its impact on the microbiome would alter the anastomotic environment and change the metastatic potential of cells. To study this, we've used two separate organoid cell lines, uh, AKPT and KPN, and these two cell lines, as well as other organoids that have been described, demonstrate a wide range of metastatic potential in animal models. However, these tend to be primary mouse models or splenic injection models, and our goal was to introduce them into a surgical model to see if manipulations of the diet or microbiome could impact their propensity for metastasis. Uh, the model we use involves giving mice 6 weeks of either a high fat diet or a normal chow diet preoperatively. We then create a clonic anastomosis in the descending colon and on postoperative day 2, we give cells an enema of either AKPT or KPN cells. We then serially sacrifice the mice, focusing mostly on postoperative day 21 and 56, and we collect samples for analysis. Our first goal was to show that these tumoroids or organizeds were able to form tumors within our model. Here you can see, uh, both on gross appearance and H&E tumors in the colon, liver, and lung, and AKPT mice with our model. We're able to confirm that these were AKPT cells by performing in situ hybridization for LGR-5, which is a clonic stem cell marker. We're able to do the same for KPN showing that both of these organoids could form tumors within our model. When we looked at tumor formation rates by 56 days, we found that there was a significantly higher rate of AKPT tumor formation with a high fat diet compared to mice that were given a chow diet. Uh, KPN mice showed no significant difference based on diet, however, interestingly, they had an opposite trend towards higher rates in the CHO diet. In addition to their higher rate of tumor formation, I noticed that AKPT mice, given a high fat diet, were often dying before their planned sacrifice day due to large bulky tumors that are found on autopsy at the anastomosis. We're able to show that these mice had significantly decreased survival compared to those on a shower diet. Interestingly, we did not see any similar trend in KPN mice, suggesting that this response to diet was at least in part based on the organized genetic background. We then wanted to test which factors within the anastomotic environment led to increased AKPT tumor formation. We hypothesized that this effect was likely mediated by the microbiome. To test this, we use fecal microbiota transplants from conventional mice to germ-free mice. We began in this experiment had a control group of germ-free mice that had not been given any FMT. So as a group that had been given FMT from high-fat diet mice. And a third group that was given an FMT from chow diet mice, and all of these mice then underwent a model and sacrificed on post-operative day 56. When isolated in a germ-free mouse model, the high fat diet associated microbiome significantly increased the rate of post-operative tumor development. In the control group and the CHO FMT group, there's a low 10 to 12% rate of tumor formation, with most of the anastomoses remaining free of tumor as shown here on the left in blue. In the high fat diet FMT group, however, there was a 40% rate of bulky tumor formation as shown in red. Having shown this impact of the high fat diet microbiota, we then wanted to test whether it could be reversed with diet manipulation. In a repeat set of experiments with conventional mice, we again had a chow diet control group, as well as a high fat diet group, and this time we introduced a reversal group in which mice were given 6 weeks of a high fat diet, followed by a chow diet reversal period preoperative. Following this diet reversal period, mice undermine our model. We began with a two-week reversal period and found that mice in the reversal group continued to have poor survival and high tumor rates similar to that of the high fat diet-fed group. We then extended this to 6 weeks, however, and the reversal group experienced a more significant increase in survival and a tumor rate similar to child fed mice, suggesting that this response to diet and its reversal of phenotype was time dependent. We then set out to analyze the stool samples from these reversal groups, beginning with 16s RNA analysis of microbiome composition. Each part here represents the complete microbiome of an individual mouse at early time points when exfoliated cells will be present within the of the bowel. You can see clearly that there's a derangement of microbiome and high fat diet mice. In particular, there's an overabundance of Alobaum shown in orange, as well as a bloom of pathogenic bacteria such as Proteus, E. coli, and Shigella on postoperative day 7, all shown in red. Uh, most importantly, however, we can see that the reversal group very closely mirrors the composition of the child group, both in its baseline composition and in its response to surgery, suggesting that the reversal in phenotype we saw may be due to changes in the microbiome. So to summarize where we are so far, uh, we're able to show that a high fat diet was associated with increased tumor burden and decreased survival for mice given AKPT cells, but not those given KPN cells. We will show with germ-free FMT experiments that the high fat diet associated microbiome was necessary for promoting higher rates of postoperative tumor formation. And lastly, with diet reversal experiments, we're able to show that preoperative diet manipulation reduces tumor formation by suggesting that there may be a clinically relevant role for diet rehabilitation and colorectal cancer treatment. I'd like to thank all of our funding sources including SSAT and ASCARS. And I'd like to thank also all the members of the Shogun Lab, as well as all of our collaborators across the University of Chicago, and that I'd be happy to answer any questions. Ryan, thank you very much, and we're gonna discuss your paper in 1 2nd. I just want to tell people that the poll from the American Hernia Society papers is now up. You can vote while we're discussing this paper. Uh, please vote. Uh, Ryan, that was a terrific paper, uh, and I have to just, uh, say that, you know, we learned in the past from the work of Turnbull and others that there were factors which affected. Uh, later metastasis, the no touch isolation technique of Turnbull was important in telling us not to handle the tumor. Uh, we know about drop metastasis and shed tumor cells when you handle, but this association with diet is very interesting. Uh, how do you suggest that we implement this in the care of our patients who are going to have colorectal surgery? Should we put them on a special diet before and for how long? Um, thank you. Uh, first of all, thank you to GlobalC for the opportunity to present this morning. Um, and it's a big question of how exactly we can translate this to, uh, some sort of clinically relevant role for diet manipulation. I think that, um, you know, it'd be difficult to compare exactly the metabolism of mice versus our colorectal cancer patients and probably a longer period than what we saw for the mice. However, um, I think it would be worth studying whether we can Uh, give patients a prescribed diet preoperatively that may improve outcomes. I, I do think that, you know, some of the data from, uh, our institution has shown that a combined uh mechanical and antibiotic preparation preoperatively is able to decrease rates of recurrence, and I think that suggests that altering that microenvironment around the anastomosis is able to impact the rates of recurrence. And I think that, uh, diet manipulation would play a role in that, uh, the exact length of time, it needs to be studied though. So do you plan to do this in humans with a prospective study? Uh, we don't have firm plans for that right now, but I, it is something that we have discussed. Should be able to do that. Doctor Rosen, any comments? Yeah, I mean, listen, first of all, Ryan, that's great work. I mean, that, that's a lot of time. That's a lot of effort. I, I actually, um, and it's well done. I mean, one of the things I love about, uh, basic science research is it's very well controlled. You have appropriate positive, negative controls, and, you know, and then you get some interesting data that you kind of try and get to figure out. So I, I, I was hoping maybe you would expand for just a minute. On, like, why would you hypothesize the difference between the AKPT and the KPN based on, you know, diet modification? Any, uh, is it just a, a, a, a model issue? I is that perhaps a barrier to then understanding it in patients? Uh, how do you kind of forecast that for your next steps? Uh, thank you. Um, so, kind of our next steps we're working on now, uh, is looking at comparing the two, organoids and how they respond to different, um, metabolites that are produced by the bacteria, and we think that that might be the link. It's explaining why there's a differential response between the two. so, uh specifically what we're looking at is, uh, secondary bile acids and deoxycholic acid metaboloids like this that are exclusively bacterially produced and What we see at least in our preliminary data in vitro is that there's a bit more of a response from the AKPT cells and the KPN cells, uh, and proliferation in response to exposure to the secondary bile acids. What that exact link is as far as pathways, that remains elusive for us. I think one thing that um yeah, we have hypothesized is it could be related to um something like wind signaling with uh the AKPT cells having uh knocked out APC signaling. Um, that's would be one difference between the two organoids, um, but we haven't exactly nailed that down yet. One of the other steps that we want to do is take the tumor samples from, uh, the mice from either organoid, and we can see with, uh, you know, RNA seq, different things like that, whether there are different pathways that are up or down regulated and if there's any differences between the two. Great stuff. All right. So, uh, I think everyone, we, not everyone voted, but we've got the, uh, a good number of people that have voted. So, uh, it looks like, um, from the final poll result that age exacerbates inequity in telemedicine one as the most impactful paper in the American Hernia Society runoff. So, congratulations to Connie. Um, you'll be going to the finals. Uh, Uh, later at the end of the hour, uh, and then we'll find out who is the best of the best. You wanna say something? No, that's, it's terrific.