Circulating immune cell populations related to primary breast cancer, surgical removal, and radiotherapy revealed by flow cytometry analysis

Abstract

Background: Advanced breast cancer (BC) impact immune cells in the blood but whether such effects may reflect the presence of early BC and its therapeutic management remains elusive.

Methods: To address this question, we used multiparametric flow cytometry to analyze circulating leukocytes in patients with early BC (n = 13) at the time of diagnosis, after surgery, and after adjuvant radiotherapy, compared to healthy individuals. Data were analyzed using a minimally supervised approach based on FlowSOM algorithm and validated manually.

Results: At the time of diagnosis, BC patients have an increased frequency of CD117⁺CD11b⁺ granulocytes, which was significantly reduced after tumor removal. Adjuvant radiotherapy increased the frequency of CD45RO⁺ memory CD4⁺ T cells and CD4⁺ regulatory T cells. FlowSOM algorithm analysis revealed several unanticipated populations, including cells negative for all markers tested, CD11b⁺CD15^low, CD3⁺CD4^-CD8^-, CD3⁺CD4⁺CD8⁺, and CD3⁺CD8⁺CD127⁺CD45RO⁺ cells, associated with BC or radiotherapy.

Conclusions: This study revealed changes in blood leukocytes associated with primary BC, surgical removal, and adjuvant radiotherapy. Specifically, it identified increased levels of CD117⁺ granulocytes, memory, and regulatory CD4⁺ T cells as potential biomarkers of BC and radiotherapy, respectively. Importantly, the study demonstrates the value of unsupervised analysis of complex flow cytometry data to unravel new cell populations of potential clinical relevance.

Keywords: Biomarker; Breast cancer; CD117; FlowJo; Granulocytes; Monocytes; Radiotherapy; Unsupervised analysis.

Fig. 1

Altered frequency of circulating monocytic populations in cancer patients. A Heat map of the FlowSOM clustering between breast cancer patients (BC) and healthy donors (HD). tSNE visualization of B the monocytic expression profile and C the differentially expressed clusters in the blood of breast cancer patients at the time of the first diagnosis vs healthy donors. Frequency of D CD117⁺ granulocytic population, and the atypical populations E 22 + 9 and F 13 + 3 at the same timing. WBC, white blood cells. Cell analysis and quantification were performed by flow cytometry with FlowJo software and results are represented as mean values +/− SD

Fig. 2

Altered frequency of circulating lymphocyte populations in cancer patients. A Heat map of the FlowSOM clustering between breast cancer patients (BC) and healthy donors (HD). tSNE visualization of B the lymphocyte expression profile and C the differentially expressed clusters in the blood breast cancer patients at the time of the first diagnosis of healthy donors. Frequency of the atypical populations D 29 + 24, E 3 + 7, and F 20 at the same timing. WBC, white blood cells. Cell analysis and quantification were performed by flow cytometry with FlowJo software and results are represented as mean values +/− SD

Fig. 3

Schematic representation of the radiotherapy study. Patients were enrolled after a confirmed histological diagnosis of breast cancer. All patients underwent conservative surgery and received standard fractionated adjuvant radiotherapy (2 Gy per session, total 50 + 10 Gy). Blood samples were collected after diagnosis was confirmed histologically, but before surgery (Sample 0), after surgery; the day of starting radiotherapy (immediately before fist irradiation, Sample 1); at the last day of radiotherapy (6 weeks after starting radiotherapy, Sample 2), and 6-8 weeks after the end of the radiotherapy (for the majority to the patients this was 12 weeks after starting radiotherapy, Sample 3)

Fig. 4

Tumor removal reduces the frequency of circulating CD117⁺ granulocytic cells. A Comparative visualization of the expression of surface markers in monocytes at different time-points of treatment by tSNE. B Heat map of the FlowSOM clustering of breast cancer patients at indicated time-points. Frequency of C monocytes and D granulocytes populations in patients at the indicated time-points during treatment. Frequency of E CD163⁺ and F CD117⁺ granulocyte population at indicated time-points relative to frequency at 0_PreOp time-point. Frequency of the G combined 16 + 18 + 21 + 12 + 15 + 26 and H 1 + 6 + 7 atypical cell populations during treatment. WBC, white blood cells. Cell analysis and quantification was performed by flow cytometry with FlowJo software and results are represented as mean values +/− SD

Fig. 5

Tumor removal and radiotherapy reduce the fraction of CD117⁺ cells within the granulocytic population. A Comparative visualization of the expression of surface markers in lymphocytes at different time-points of treatment by tSNE. B Heatmap of the FlowSOM clustering of breast cancer patients at indicated time-points during treatment. Frequency of the atypical populations C 25, D 41, and E 23 + 30 in patients during treatment. Relative quantification to 0_PreOp time-point of F CD4+ CD45RO+ lymphocytes and G CD45RO+ regulatory T cells during treatment. WBC, white blood cells. Cell analysis and quantification was performed by flow cytometry with FlowJo software and results are represented as mean values +/− SD