# Microbial and Immune Landscape of Malignant Ascites: Insights from Gut, Bladder, and Ascitic Fluid Analyses

## TL;DR

This study investigates the role of gut and bladder microbes and immune cells in malignant ascites, finding limited bacterial presence and a suppressed immune response in the fluid.

## Contribution

The study reveals specific bacterial taxa linked to peritoneal metastasis and highlights an immunosuppressive environment in malignant ascites.

## Key findings

- Malignant ascites contains very few bacteria, making microbiome analysis difficult.
- Certain bacterial groups like Clostridia and Gammaproteobacteria are associated with peritoneal metastasis.
- Immune cell populations in malignant ascites show suppression, particularly in T and NK cells.

## Abstract

Malignant ascites, a fluid buildup in the abdomen due to advanced cancer, is associated with poor prognosis; yet, its underlying causes remain unclear. This study explores whether microbes from the gut and bladder contribute to malignant ascites and examines the immune environment within the fluid. Using genetic sequencing and immune cell analysis, we found that malignant ascites contains very few bacteria, making it difficult to assess microbial influences. While overall gut and bladder microbiomes showed no significant changes, specific bacterial groups were linked to peritoneal metastasis. Additionally, immune cell populations in malignant ascites suggested a suppressed immune response. These findings highlight the complexity of malignant ascites and suggest that larger studies using advanced techniques may be needed to better understand its development and potential treatment strategies.

Background/Objectives: Malignant ascites frequently arises in advanced cancers with peritoneal metastasis and is associated with poor outcomes. Known mechanisms include lymphatic obstruction by tumor cells, increased vascular permeability, and sodium retention via the renin–angiotensin–aldosterone system; however, the pathogenesis remains not fully understood. We investigated whether gut and bladder microbiomes correlate with malignant ascites development or progression and whether the immune microenvironment in ascitic fluid is altered. Methods: We enrolled 66 histologically confirmed cancer patients, dividing them into malignant ascites (n = 20) and non-ascites (n = 46) groups. Stool, urine, and ascitic fluid samples were analyzed using 16S rRNA next-generation sequencing. Immune cell subsets in ascitic fluid were characterized using flow cytometry. Results: In 19 of the 20 malignant ascites samples, the bacterial load was too low for reliable 16S rRNA sequencing, suggesting that malignant ascites is largely sterile. The overall gut microbiome diversity did not differ significantly by ascites status, although a trend emerged in patients with peritoneal metastasis, including the enrichment of class Clostridia and Gammaproteobacteria. Bladder microbiome analysis also showed no significant differences in ascites or metastasis status. Flow cytometry revealed reduced T-cell (CD3+, CD4+, CD8+) and NK cell (CD56+) populations compared to data from cirrhotic ascites. Conclusions: Malignant ascites exhibit minimal bacterial biomass, making comprehensive microbiome analysis challenging. Although no major global changes were noted in gut and bladder microbiomes, specific taxa were linked to peritoneal metastasis. These findings highlight an immunosuppressive ascitic environment and suggest that larger-scale or multi-omics approaches may help elucidate the role of microbiota in malignant ascites.