Banerjee, Narendra

Abstract Mesenchymal stem cell (MSC)-derived exosomes are recognized as critical mediators within the tumor microenvironment (TME), exerting both pro- and anti-tumorigenic effects depending on contextual factors. These vesicles are also gaining attention for their potential as therapeutic vehicles in regenerative medicine and targeted drug delivery. However, the influence of the TME on the physical characteristics of MSC-derived exosomes remains poorly understood. In this study, we utilized Atomic Force Microscopy (AFM) to investigate the morphological and nanomechanical properties of MSC-derived exosomes under standard and TME-like conditions. AFM imaging in fluid mode preserved the native structure of exosomes and enabled high-resolution analysis of their topography, surface roughness, stiffness, adhesion, and deformation. AFM offers unique advantages in exosome research by enabling label-free, nanoscale analysis of vesicle properties in near-physiological conditions. The ability to detect such subtle but functionally significant changes highlights the relevance of AFM in exosome characterization and quality assessment. Our results revealed that exposure to the TME induces marked changes in exosomal membrane morphology and mechanical behavior, including increased surface heterogeneity, higher stiffness, and altered adhesive interactions. These biophysical alterations may reflect changes in membrane composition and protein or lipid cargo, potentially affecting exosome function, uptake, and therapeutic efficacy. Overall, our findings provide new insights into how the TME modulates MSC exosome biophysics and underscore the utility of AFM-based techniques for advancing the development of exosome-based diagnostics and therapies. This can provide a conceptual framework for future mechanistic and translational studies in this area.

Pancreatic cancer ranks third in cancer related deaths in the USA, however, no reliable theragnostic biomarker exists for the disease. The community of bacteria within the body can become dysbiotic and promote cancer through inflammation and other means. The objectives of this study were to determine the identity and abundance of bacterial species in the gut of mice before and after development of pancreatic cancer. As there can be significant variation in the genomes of different strains of bacterial species, a metagenome analysis was also performed. We hypothesized that there would be significant variation in species and genes of the gastrointestinal tract of mice before and after xenografting with pancreatic cancer. A decrease in ecological diversity was also predicted. Fifteen female SCID mice were divided into three cages. To develop the SubQ pancreatic cancer model, 5 X 106 Panc1 cells were injected at the flank. Fecal materials were collected prior to tumor cell implantation and after tumor development. DNA was extracted from the fecal material. For phylogenetic analysis the seven variable regions of the 16S rRNA gene were amplified and sequenced. Metagenomic analysis was performed as well. Average tumor size in mice after 40 days was ∼ 32 mm3. Microbial diversity was decreased in two out of the three groups of mice. Principle component analysis showed major differences between all samples. There were at least 15 OTUs in each set of samples specific to pretumor mice and 15 OTUs specific to tumor bearing mice. One genus of bacteria, Alistipes, was specific to each set of tumor bearing mice. Taxa at significantly increased abundances in tumor bearing mice included Prevotellaceae and Porpyromonadaceae. Moreover, there were several gene functions that were enriched in tumor bearing mice. This study produced data that demonstrates that there is a link between health status and the gut microbiome. However, this is not a new idea. This study could serve as foundation to establish a gut microbiota profile of pancreatic cancer. Precision medicine is becoming more common due to the decreasing costs of DNA sequencing. The findings of our investigation strengthens the hypothesis that there is an interaction between gut microbiome and pancreatic cancer development and bacteria or bacterial genes could serve as theragnostic biomarker for pancreatic cancer. Acknowledgement: This research was supported by NIH Grant# T34-GM100831, NSF-NOYCE Graduate student training award, a US Department of Education Graduate student training award to Elizabeth City State University Campus of The University of North Carolina and a University of North Carolina Collaboratory Grant award to Dr. H. Banerjee and Dr. Colby Hunter. Citation Format: Gabrielle Morgan, Colby Hunter, Michael Hall, Narendra Banerjee, Erik Armstrong, Kanisha McNeil, Karington Perry, Santanu Bhattacharya, Hirendra N. Banerjee, Krishna K. Rachamala, Shamit Dutta, Naga M. Nakka. Phylogenetic and metagenomic analysis of the human pancreatic cancer microbiome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6526.

Abstract Pancreatic cancer is a significant public health concern, with increasing incidence rates and limited treatment options. Recent studies have highlighted the role of the human microbiome, particularly the gut microbiota, in the development and progression of this disease. Microbial dysbiosis, characterized by alterations in the composition and function of the gut microbiota, has been implicated in pancreatic carcinogenesis through mechanisms involving chronic inflammation, immune dysregulation, and metabolic disturbances. Researchers have identified specific microbial signatures associated with pancreatic cancer, offering potential biomarkers for early detection and prognostication. By leveraging advanced sequencing and bioinformatics tools, scientists have delineated differences in the gut microbiota between pancreatic cancer patients and healthy individuals, providing insights into disease pathogenesis and potential diagnostic strategies. Moreover, the microbiome holds promise as a therapeutic target in pancreatic cancer treatment. Interventions aimed at modulating the microbiome, such as probiotics, prebiotics, and fecal microbiota transplantation, have demonstrated potential in enhancing the efficacy of existing cancer therapies, including chemotherapy and immunotherapy. These approaches can influence immune responses, alter tumor microenvironments, and sensitize tumors to treatment, offering new avenues for improving patient outcomes and overcoming therapeutic resistance. Overall, understanding the complex interplay between the microbiome and pancreatic cancer is crucial for advancing our knowledge of disease mechanisms and identifying innovative therapeutic strategies. Here we report phylogenetic analysis of the 16S microbial sequences of the pancreatic cancer mice microbiome and corresponding age matched healthy mice microbiome. We successfully identified differentially abundance of microbiota in the pancreatic cancer.

Abstract The microbiome refers to the diverse community of microorganisms, including bacteria, viruses, fungi, and other microbes, that inhabit various parts of the human body, such as the skin, mouth, gut, and other mucosal surfaces. The role of the microbiome in cancer is a complex and evolving area of research. While much is still being learned, there is evidence suggesting that the microbiome can influence cancer development and progression in several ways. Some bacteria within the microbiome can produce substances that may be either carcinogenic or protective against cancer. For instance, the conversion of certain dietary components by gut bacteria can produce metabolites that may either promote or inhibit cancer development. Different cancers may be associated with specific alterations in the microbiome, for example, the gut microbiome has been extensively studied in relation to colorectal cancer, with certain bacterial species potentially contributing to the development of this cancer. However, the role of microbiome in Pancreatic cancer is still not extensively studied, henceforth, we did a preliminary study to identify novel bacterial communities around pancreatic cancer microbiome by 16SrDNA sequencing in human pancreatic cancer xenograft model mice. Isolating total genomic DNA from the pancreatic model mice feces, 16SrDNA PCR was done using specific primers and the amplicon obtained as PCR product was confirmed by standard DNA gel electrophoresis with a 1 kb standard ladder to be 1.5kb in size universal for all bacterial 16SrDNA. The amplicon was then sent for Next Generation Sequencing at Zymo Research Corporation (CA). The resultant DNA sequences were analyzed by bioinformatics studies and their phylogeny identified by comparing with 16SrDNA data base. Differential abundance of the bacteria’s of the genera Lachnoclostridium, Robinsoniella, Alistipes, Akkermansia and Lactobacillus were observed analyzing the data along with other bacterial communities. We are currently continuing the project to identify more bacterial communities of significance in the pancreatic cancer microbiome and investigating the role and significance of these bacterial communities in pancreatic cancer tumor microenvironment. Acknowledgement This study was done by a joint collaboration between scientists and students of Elizabeth City State University (ECSU) campus of the University of North Carolina Systems and Mayo Clinic, Jacksonville. This research is supported by the NIH-NIGMS grant#T34GM100831-10 to Dr. H. Banerjee and a University of North Carolina General Administration Grant to Dr. Colby Hunter at ECSU. Citation Format: Hirendra N. Banerjee, Colby Hunter, Narendra Banerjee, Karrington Perry, Khadimou Dia, Anasua Banerjee, Santanu Bhattacharya, Hari Krishnareddy Rachamala, Enfang Wang, Shamit Dutta. An investigation to identify the phylogeny of bacterial communities in the pancreatic cancer microbiome by 16SrDNA sequencing and bioinformatic analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2795.

Abstract Cancer is a public health crisis affecting approximately 19.3 million globally, annually. Lung cancer has the highest percentage of incidence and is responsible for 27% of cancer-related deaths. The prevalence of lung cancer worldwide led us to investigate differential gene expression in lung adenocarcinoma and small cell lung cancer cell lines in comparison to a healthy lung cell line. We also investigated the effect of lung tumor microenvironment to bone marrow derived pluripotent mesenchymal stem cells (PMSC) and tumor associated macrophages. This research involved cancer and healthy cell line culture with appropriate medium. Whole transcriptomic and small non-coding RNA sequencing and analysis by Ingenuity Pathway Analysis (Qiagen, USA), Quantitative RT-PCR and ELISA assays. We further studied morphological changes and differential gene expression in extracellular vesicles(exosomes) isolated from the TAM and TME exposed PMCs by atomic force microscopy and RNA sequencing techniques. The results of our investigation showed differential gene expression and novel core canonical pathways in the lung cancer cells, TME exposed TAM, and PMSC. We also observed morphological changes in TME derived exosomes from TAM and PMSC. RNA sequencing results showed differential gene expression of both coding and non-coding RNAs including tRNA fragments. Thus, our findings of this investigation led to identification of multiple cancer related genes and small non-coding microRNA, long non-coding RNA, and tRNA fragments which will have potential diagnostic and therapeutic target value that demands further future research. Acknowledgement: This research is supported by a grant from US NIH-NIGMS- 5T34GM100831-08 and US-NIH Grant# U 54 MD007597-31-5959. Citation Format: Hirendra N. Banerjee, Joseph Hedley, Cheslsey Aurelus, Narendra Banerjee, Kuldeep Rawat, Quentin Reaves, Shennel Brown, Darla Gilmartin, Makieyah Liverman, Jazmine Cuffee, Elizabeth Cagle, Erik Armstrong, Brent Lake, Santosh Mandal, Somiranjan Ghosh, Stephen Beebe, Kamal Asadipour, Christopher Krauss, Santanu Bhattacharya, Tanmay Kulkarni, Zahidur Abedin. An investigation to study differential gene expression of lung cancer cells, tumor associated macrophages, and bone marrow derived mesenchymal stems cells in the tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5477.