Period From 1/2022 Through 6/2022
The group held conference calls on February18, April 22, and June 17. The next conference call is scheduled for August 12.
Feb. 18. Dr. Testerman reported that she and her collaborator, Dr. Jie Li, have just submitted a new proposal to NORD to continue their collaboration on PMP. Dr. Lie has been working with Dr. Testerman and purifying compounds from the PMP cell cultures that Dr. Testerman has grown. Since Dr. Testerman will be staying in the area, she can continue her interaction with Dr. Lie on the PMP research. They have a lot of in vitro data on one interesting compound that they plan to study further in vivo. They also plan to study a number of other compounds in vitro. For the in vivo work, they will look into growing subcutaneous tumors. Hydrogel will also be studied for its ability to help tumor material grasp tissue and thereby facilitate tumor growth. For example, tumor material with and without hydrogel can be implanted on either side of an animal and the growth of the 2 tumors compared. It is planned to test the interesting compound in vivo to determine its effect on the growth of PMP tumors. Dr. Testerman and Dr. Lie have found that some of the PMP cells that are cultured live only for a couple of generations and then die. To overcome this problem, they also plan to transect cells with cagA from Helicobacter pylori in order to try to keep them growing. The group expressed their gratitude to Dr. Testerman for her dedication and continued efforts on researching PMP.
Dr. Metcalf mentioned that she and Victoria had just received a grant to study tumor microbiome. The grant came from an internal program at Colorado State University and Colorado University, called the Colorado Clinical and Translational Sciences Institute (CCTSI), which is funded by NIH. The grant involves work with a canine model and Ms. Nieciecki has found 1 paper that discusses improved amplification in a canine model. Ms. Nieciecki discussed her work on the 3 PMP samples that she has been sequencing. PMP433 has more bacteria in it than the other two samples. In all her sequencing results Ms. Nieciecki has noted a number of off-target results. The off-target results come from human DNA present in the PMP tumor specimens. The differences she is seeing in the amount of bacteria in samples indicate that PMP tumors have varying bacterial loads. Dr. Merrell noted that even cases where there are few reads can yield useful information. A discussion took place about what primers to use and whether longer primers might work better. Also, how to approach the sequencing of the PMP samples in general was discussed. Whether the research will yield a “core” microbiome was discussed as was the issue of whether we would be able to understand the mechanistic aspects of the effects of bacteria on PMP tumor growth.
During the last conference call Drs. Merrell, Johnson and Metcalf and Ms. Nieciecki agreed to hold a conference call in January to discuss how to proceed with writing up a manuscript for what we have found out from our microbiome sequencing studies. They discussed the next steps involved with writing up a manuscript, which will be done once new data are available.
April 22: Dr. Li reported that he has been collaborating with Dr. Testerman on her PMP research for more than 2 years. Dr. Li is in the Department of Chemistry and Biochemistry at the University of South Carolina, which he joined in 2019. He is an Assistant Professor with research interests in bioorganic chemistry, drug discovery, genome mining, biosynthesis, and biosynthetic engineering. Dr. Li has had a student working in Dr. Testerman’s lab and the team has identified functional metabolites from bacterial cultures. The student is studying the chemistry and biochemistry of the metabolites. They have confirmed that some of the compounds that have been isolated affect cancer cells. Dr. Li has recently been notified that he has been awarded a NORD grant to continue studies on PMP that were initiated by Dr. Testerman. Dr. Li mentioned that he and Dr. Testerman submitted one proposal on their research to NIH but it was not funded. He plans to continue the culturing work that was initiated by Dr. Testerman. It was mentioned that Dr. Li would have to be added to the material transfer agreement that Dr. Testerman had with Mercy Medical so that he can receive tumor specimens from Mercy. He will likely also need to be added to the IRB approvals.
Ms. Nieciecki gave an update on her microbiome research on the PMP samples she has studied. As discussed at the last meeting, she will move forward with sequencing all of the samples sent to her from USU. To this end, she has now isolated DNA from the 26 tumor specimens and she is in the process of PCR amplifying the 16S gene and sending these amplicons for sequencing. She hopes to have the sequencing done by the end of May. Her goal was to try to corroborate the results that Dr. Johnson got earlier. Ms. Nieciecki mentioned the difficulty of working with PMP tumors where there is a low microbiome content (low biomass), which can lead to amplification problems because of the large amount of host cell DNA that is present. A long discussion on issues associated with amplifying bacterial DNA from tumors then took place.
Ms. King said that there was not much new that could be reported from Mercy. Four new PMP patients will undergo surgery. This situation at Mercy has been somewhat hectic lately. The practice manager and surgical coordinator resigned, and 2 new fellows and 3 interns started. Since Michelle left, the other research coordinator position has not been filled as yet. Ms. King is still working on getting the fecal microbiome patients who have not completed their paperwork to do so. Nothing has been sent/collated yet. One of our interns did however submit a paper to Current Oncology for a special PMP issue, reviewing the current available data on microbiome in appendiceal cancer. The paper was submitted at the end of March and hopefully Mercy will hear something back in the next couple weeks.
June 17: Dr. Li reported that he has been continuing his research with Dr. Testerman on studying bacterial metabolites in the cultures developed by Dr. Testerman. The South Carolina team was awarded a new 2-year NORD grant for $50000 to study “Improving PMP models for pre-clinical testing and evaluating PMP associated bacterial functional metabolites in PMP progression and treatment”. Dr. Li said that he will be contacting Mercy to work out the details for a material transfer agreement with them.
Ms. Nieciecki gave an update on her microbiome research at Colorado State University using a Powerpoint presentation. Her overall goal is to confidently identify bacterial taxa associated with PMP tumors. To do so Ms. Nieciecki is evaluating and removing contaminant taxa; determining if tumor samples are similar between extraction locations; and trying to identify core PMP taxa. She used Dada2 denoising followed by taxonomy filtering to remove unassigned, mitochondria, chloroplast, eukaryota, and mammalia reads. Negative extraction controls appear to cluster with tissue sample, suggesting tissue and negative communities are similar to each other possibly due to contamination. Ms. Nieciecki discussed her contamination results and provided a detailed comparison between results achieved by her and by Dr. Johnson. After decontamination Ms. Nieciecki noted that samples appear to cluster depending on the extraction location. Some samples shared the same taxonomy but others differed depending on extraction location. Lastly, Ms. Nieciecki gave a list of the top 25 ASV’s that she found in the decontaminated tumor dataset. Dr. Metcalf suggested that samples having low reads may be dominated by contaminants. Dr. Metcalf also suggested that there might be a transient microbiome in patients depending on their state just prior to surgery, e.g. stress. Such transient phenomena could confound an attempt to identify a core PMP microbiome.
In 2016 Dr. McAvoy heard a presentation on using HIPEC to treat ovarian cancer (OC) and the fact that it seemed to improve surgical results suggested that bacteria might be involved in OC. Dr. McAvoy felt that getting funding for our research would be much easier for studying OC compared to PMP. In 2016 he searched the NIH PubMed website and found only 2 papers on bacteria and OC, while his recent search yielded 114 papers. Prior to the conference call Dr. McAvoy emailed a list of 10 papers including abstracts that he found on the NIH PubMed website. The first 8 papers indicate:
- That bacteria definitely have been found in OC samples.
- That the microbiome of OC patients is more diverse than the microbiome of healthy females.
- That the microbiome difference between OC patients and healthy controls can used for early disease detection which can be very beneficial since in its early stages OC does not exhibit symptoms.
- That the OC microbiome can be used to predict whether an OC patient will be resistant to chemotherapy.
One of the 8 papers discussed combining OC patient genetic information with their microbiome results for prediction. One question that remains to be answered is whether bacteria cause OC or they take advantage of the disease. The OC microbiome papers in general discuss interesting approaches for applying microbiome results to treat an important cancer. The last 2 papers that Dr. McAvoy found interesting discussed:
- Using machine learning on microbiome results to separate cancer and non-cancer patients
- Using bacterial DNA circulating in blood for correlation with different types of cancer.
A discussion about doing research on the OC microbiome took place.
The group held conference calls on Aug. 12, Oct. 7, and Nov. 18. The next conference call is scheduled for Jan. 13, 2023.
Aug. 12: Ms. Nieciecki gave an update on her microbiome research using a Powerpoint presentation. Ms. Nieciecki has been studying both diversity metrics as well as decontamination issues for her samples. She is considering using quality metrics and if these are not met she would remove those samples from the analysis. For example, if the negative extraction control contributes greater than 20% to a sample’s sequence, then the sample would be dropped. Another metric involves dropping a sample if it does not generate enough reads. Ms. Nieciecki presented a slide that showed ASV overlaps between her sequencing and that done by Dr. Johnson. Many of the samples had 2 or in some cases more overlapping ASV’s. Ms. Nieciecki speculated that if the tumor samples were heterogeneous then this could lead to a small number of overlaps. She discussed the possibility of sequencing multiple sub-samples from a specimen to overcome this problem. A discussion took place about using fluorescence in situ hybridization (FISH) on samples to assess overall homogeneity. Ms. Nieciecki said that she would check to see if there is a group at Colorado State that is proficient in doing FISH analysis. Dr. Merrell suggested that Ms. Nieciecki take a look at our earlier paper with Jeremy Galbraith as the lead author since it discusses the FISH protocol that we used in earlier research. A discussion was also held on whether tumor samples could be homogenized prior to sequencing and potentially compared with non-homogenized samples in terms of the sequences produced.
Oct. 7: Dr. Li is preparing a manuscript on his research with Dr. Testerman that involves isolating functional metabolites from PMP bacterial strains that Dr. Testerman isolated. Dr. Li has studied the activity of these metabolites in regard to their ability to affect tumor growth in cancer cell lines. Li discussed the cancer cell lines he has tested and the various assays that he has used.
Ms. Nieciecki gave an update on her microbiome research using a Powerpoint presentation. Ms. Nieciecki has been studying the problem of how to accurately identify bacteria found in PMP samples via microbiome analysis. Since some PMP samples have a small quantity of bacteria, Ms. Nieciecki is developing a quality protocol to determine whether a sample gives a robust bacterial signal. Ms. Nieciecki presented the results that she has gotten to date on PMP samples. Additional open issues that need to be addressed were discussed.
Nov. 18: Dr. Li did not have any new results to report. He is preparing a manuscript on his research with Dr. Testerman that involves isolating functional metabolites from PMP bacterial strains that Dr. Testerman cultured. The NORD grant that Dr. Li received has an animal component in it. He plans to use Dr. Testerman’s animal model and protocol when he carries out additional experiments. Dr. Merrill raised the issue that Dr. Li needs to be added to the IRB protocol with Mercy Medical so that tumor samples can be sent to him. Additionally, since he now has possession of prior samples sent to Dr. Testerman, he needs to be on the protocol. A discussion took place about adding Dr. Li to the protocol. Ms. King said that adding him should not be difficult at all.
Dr. Metcalf discussed the manuscript that she and Ms. Nieciecki are preparing. They had sent some samples to a collaborator to see if he could image the bacteria in the samples. The collaborator used FISH and another type of imaging but was not successful. Given the negative imaging results, Dr. Metcalf raised the question of whether we should pursue submitting a manuscript on the microbiome research. She pointed out that only 4 samples had been studied for imaging and that only a few slices of tissue/sample were available. She felt that even though the imaging was negative, we should move forward with the paper. A discussion took place and several group members felt that the negative results were not uncommon. Ms. Nieciecki mentioned that she had recently seen 2 new papers that discussed the fact that tumor samples could be very heterogeneous and thereby not allow positive images to be obtained. The papers dealt with oral and colorectal cancer. During the discussion it was pointed out that bacteria can be detected by PCR even when they do not show up in imaging. Dr. Metcalf said that we could have a section in the paper that pointed out that we will look into imaging in the future when more samples than the 4 that were studied could be analyzed. The group agreed that we should move ahead with the paper. Dr. Metcalf would like to have a draft submitted before Christmas.
Ms. King introduced Ms. Katie Pawlikowski who has recently joined Mercy as a research coordinator. Ms. Pawlikowski will take over the fecal microbiome project and Dr. Metcalf offered to help if any problem with communicating with the University of San Diego arose. Ms. King mentioned that a marketing coordinator at Mercy had alerted her to a press release from Dr. Lowy stating that PMP could not be studied in mouse models. Dr. Testerman discussed why, based on her extensive mouse research, she disagreed with this statement. Dr. Merrell asked Ms. King to circulate the announcement to the group.