Metagenomic Analyses Reveal Distinct Gut Microbiota Signature for Predicting the Neoadjuvant Chemotherapy Responsiveness in Breast Cancer Patients

Abstract

Background: Growing evidence supports the modulatory role of human gut microbiome on neoadjuvant chemotherapy (NAC) efficacy. However, the relationships among the gut microbiome, tumor-infiltrating lymphocytes (TILs), and NAC response for breast cancer (BC) patients remain unclear. We thus proposed this preliminary study to investigate the relationship between gut microbiome and BC patients' responses to NAC treatment as well as underlying mechanisms.

Methods: Prior to receiving NAC, the fecal metagenome collected from 23 patients with invasive BC was analyzed. Patients were subsequently assigned to the NAC non-effectual group and the NAC effectual group based on their response to NAC. The peripheral T lymphocyte subset counts were examined by flow cytometry methods. CellMinor analysis was employed to explore the relationship between CD4 mRNA expression and the reaction of tumor cells to NAC drugs.

Results: The gut microbiomes of the NAC non-effectual group showed characteristics of low diversity with low abundances, distinct metagenomic composition with decreased butyrate-producing and indolepropionic acid-producing bacteria, and increased potential pathobionts compared with the NAC effectual group. The combination of Coprococcus, Dorea, and uncultured Ruminococcus sp. serves as signature bacteria for distinguishing NAC non-effectual group patients from the NAC effectual group. The absolute numbers of CD4⁺ and CD8⁺ TIL infiltration in tumors in the NAC non-effectual group were significantly lower than those in the effectual group. Similar findings were reported for the CD4⁺ T lymphocytes in the peripheral blood (p's < 0.05). NAC effectual-related signature bacteria were proportional to these patients' CD4⁺ T lymphocyte counts in peripheral blood and tumors (p's < 0.05). CellMinor analysis showed that the CD4 mRNA expression level dramatically climbed with increased sensitivity of tumor cells to NAC drugs such as cyclophosphamide, cisplatin, and carboplatin (p's < 0.05).

Conclusions: The composition of the gut microbial community differs between BC patients for whom NAC is effective to those that are treatment resistant. The modulation of the gut microbiota on host CD4⁺ T lymphocytes may be one critical mechanism underlying chemosensitivity and NAC pathologic response. Taken together, gut microbiota may serve as a potential biomarker for NAC response, which sheds light on novel intervention targets in the treatment of NAC non-effectual BC patients.

Keywords: CD4+ T lymphocytes; breast cancer; gut microbiota; neoadjuvant chemotherapy; pathologic response.

Figure 1

The recruitment of participants and the process of sample collection.

Figure 2

Comparisons of alpha-diversity and beta-diversity between the NAC effectual group (n = 5) and the NAC non-effectual group (n = 18). (A–C) Alpha-diversity of the two groups at the species level, measured in terms of the Shannon (A), Simpson (B), and Chao1 index (C). (D) PCoA of unweighted UniFrac analysis showed that the overall fecal microbiota composition was different between the NAC non-effectual group and the NAC effectual group. NAC, neoadjuvant chemotherapy; PCoA, principal coordinate analysis. Superscript symbols indicate statistically significant differences between the two groups: *p < 0.05, **p < 0.01.

Figure 3

Relative abundance of 75 species differing significantly between the NAC non-effectual group and the NAC effectual group (LDA effect size analysis).

Figure 4

Disease classification based on gut microbiome signature. (A) Classification performance of the random forest model using the relative abundance of NAC- efficacy associated genera was assessed by the area under the ROC in BC patients. The combination of 3 signature bacteria: Coprococcus, Dorea, and uncultured Ruminococcus sp. The combination of 9 optimal species markers: Bacteroides, Coprococcus, Dorea, Fusicatenibacter, Ruminococcus, Butyrivibrio sp. CAG 318, Lactobacillus salivarius, Bacteroides xylanisolvens, and uncultured Ruminococcus sp. (B) Identification of the signature gut microbiota associated with NAC- efficacy by random forest. Fivefold cross-validation together with random forest was performed to determine the signature biomarkers. Detailed signature biomarkers’ random seed from the random forest is presented between the NAC non-effectual group and the NAC effectual group.

Figure 5

(A) The presence and abundance of tumor-infiltrating CD4⁺ TILs as assessed with IHC staining between representing NAC non-effectual patients (P1, P2, P3) and NAC effectual patients (P4, P5, P6). Scale bars, 50 μm. (B) The presence and abundance of tumor-infiltrating CD8⁺ TILs as assessed with IHC staining between representing NAC non-effectual patients (L1, L2, L3) and NAC effectual patients (L4, L5, L6). Scale bars, 50 μm. IHC, immunohistochemistry. Data show means ± s.d. ** denotes p < 0.01 by Student’s t-test. *** denotes p < 0.001 by Student’s t test.

Figure 6

Correlation between relative abundance of signature gut microbiota and T lymphocyte cell subsets in breast cancer patients treated by NAC. Partial Spearman’s rank correlation coefficient is indicated using a color gradient: red indicates positive correlation; blue, negative correlation. TIL, tumor infiltrating lymphocyte. * denotes p < 0.05.

Figure 7

CD4 mRNA and CD8 mRNA expression level is related to the sensitivity of tumor cells to NAC drugs. (A–C) CD4 mRNA expression level is significantly and positively correlated with the sensitivity of tumor cells to (A) cyclophosphamide (r = 0.81, p < 0.0001), (B) cisplatin (r = 0.30, p < 0.05), and (C) carboplatin (r = 0.47, p < 0.001). (D) CD8 mRNA expression level is significantly and positively correlated with the sensitivity of tumor cells to cyclophosphamide (r = 0.41, p < 0.01). (E, F) CD8 mRNA expression level is not significantly correlated with the sensitivity of tumor cells to (E) cisplatin (r = 0.18, p = 0.18) and (F) carboplatin (r = 0.18, p = 0.17).

Figure 8

An overview of the microbiota–T lymphocyte (TILs) interactions that modulate neoadjuvant chemotherapy (NAC) efficacy. NAC treatment induces changes in the gut microbiota diversity, causing inflammation and damage to the mucosal barrier of cancer patients, permitting pathogenic bacteria to cross the intestinal barrier and enter lymphoid organs. Then the intestinal microbiota may mediate the induction of TILs such as CD3⁺, CD4⁺, and CD8⁺ cells in patients undergoing NAC. When the tight junctions between epithelial cells are broken and the intestinal permeability reduces, pathogenic bacteria interact with immune cells, regulating the response rate of the NAC treatment.