We have previously demonstrated that histamine produces a complex regulation of breast cancer (BC) immunobiology. Increasing evidence suggests that tumor microbiome impacts both the progression of BC as well as treatment’s efficacy. In this study, we aimed to characterize the intratumor microbiota of the translationally relevant murine 4T1 BC model and to reveal whether it can be modified by histamine treatment.
We developed a syngeneic model by orthotopic inoculation of 4T1 cells in Balb/c mice, which were treated daily with histamine (s.c. 10 mg/kg) or left untreated. After 3 weeks, tumors were obtained, and DNA was extracted to analyze the bacterial composition by sequencing the hypervariable regions V3-V4 of 16S rRNA gene. Bioinformatics analysis was performed with QUIME 2 (v 2022.2.1).
Histamine reduced tumor growth and vascularization while increasing tumor apoptosis. The taxonomic analysis showed a predominance of phylum Proteobacteria (57.5 % of the overall reads sequenced), Firmicutes (13.3 %), and Actinobacteria (10.4 %) in untreated mice, which was like those reported in human BC. Bacteroidetes (2.5%) and Cyanobacteria (1.5%) were detected to a lesser extent. Two relevant genera found were Pseudomonas (Proteobacteria; 6.4 %) and Streptococcus (Firmicutes; 3.5 %). Preliminary studies showed that histamine produced changes in the distribution of order-level phylotypes, significantly decreasing Proteobacteria (48.2%), Actinobacteria (3.8%), Cyanobacteria (0.3%) while increasing Firmicutes/Bacteroidetes (F/B) ratio (7.4 vs. 5.6). 
We conclude that histamine favorably modified the intratumor microbiota by reducing phyla associated with breast cancer risk and improving F/B. It also reduces Pseudomonas and Streptococcus genera both related to tumor development. However, the causal relationship between the microbiota and histamine-induced proliferation inhibition needs to be directly tested in future experiments.