Monitoring the responsiveness of T and antigen presenting cell compartments in breast cancer patients is useful to predict clinical tumor response to neoadjuvant chemotherapy

Abstract

Background: Vaccination of mice with tumors treated with Doxorubicin promotes a T cell immunity that relies on dendritic cell (DC) activation and is responsible for tumor control in vaccinated animals. Despite Doxorubicin in combination with Cyclophosphamide (A/C) is widely used to treat breast cancer patients, the stimulating effect of A/C on T and APC compartments and its correlation with patient's clinical response remains to be proved.

Methods: In this prospective study, we designed an in vitro system to monitor various immunological readouts in PBMCs obtained from a total of 17 breast cancer patients before, and after neoadjuvant anti-tumor therapy with A/C.

Results: The results show that before treatment, T cells and DCs, exhibit a marked unresponsiveness to in vitro stimulus: whereas T cells exhibit poor TCR internalization and limited expression of CD154 in response to anti-CD3/CD28/CD2 stimulation, DCs secrete low levels of IL-12p70 and limited CD83 expression in response to pro-inflammatory cytokines. Notably, after treatment the responsiveness of T and APC compartments was recovered, and furthermore, this recovery correlated with patients' residual cancer burden stage.

Conclusions: Our results let us to argue that the model used here to monitor the T and APC compartments is suitable to survey the recovery of immune surveillance and to predict tumor response during A/C chemotherapy.

Keywords: Breast cancer; Chemotherapy; Dendritic cells; Doxorubicin; Immune-monitoring; Neoadjuvant; T cells.

Conflict of interest statement

Ethical approval and consent to participate

This study was approved by the ethics committee of the Instituto Nacional de Cancerología (Bogotá-Colombia) by the reference number ACT-018 May 2012. The healthy donors and breast cancer patients sign the informed consent before the blood sample was drawn.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1

Assessing different cell populations ex vivo in PBMCs from healthy donors and BC patients before and after chemotherapy. a Paired analysis of tumor size (area in cm2) of the patients before therapy and after three cycles of A/C chemotherapy (n = 17). b Working strategy for multi-parametric cell analysis using flow cytometry. Monocytes and lymphocytes were defined by contour plots using SSC-A vs. FSC-A. Myeloid and plasmacytoid dendritic cell (DCs) (cells HLA-DR+ Lin1/CD15- CD11c + or HLA-DR+ Lin1/CD15- CD123+ respectively) and myeloid-derived suppressor cells (MDSCs) (cells HLA-DR- Lin1/CD15- CD13+ CD33+ ± Arginase 1+), were analyzed within the monocytic cell region. Finally, the percentage of CD4+ and regulatory T cells CD4+ CD25+ CD127- FoxP3+ (Tregs) was estimated within the lymphoid cell region. c Percentage of different sub-populations ex vivo in PBMCs from healthy donors (white box n = 10) and BC patients before (gray box n = 12) and after chemotherapy (dashed box n = 12). Panels summarize the percentages of DCs populations (top panel), MDSCs (middle panel) and CD4+ and CD4+ Tregs: CD4+ CD25+ CD127- and CD4+ CD25+ CD127- FoxP3+ (middle and right panels at the bottom). Paired analysis by Wilcoxon test, *** p < 0.001. Box and whiskers graph with 10–90% of data

Fig. 2

DC maturation and IL-12p70 production are hampered in cancer patients before treatment. a The analysis by contour plots of a representative sample of myeloid (HLA-DR+ Lin1- CD11c+) and plasmacytoid (HLA-DR+ Lin1- CD123+) DCs is shown. b Representative histograms comparing the phenotype (CD83, CCR7, and CD86) of immature (empty histogram) and mature (gray histogram) in myeloid DCs (HLA-DR+ Lin1- CD11c+) derived from HD. c Quantification of CD83 expression in response to maturation stimulus (delta of the percentage of CD83 expression between mature and immature DCs) in DCs derived in PBMCs from HD (white box), and breast cancer patients before and after chemotherapy (grey and dashed box, respectively) in monocytic cells (defined by FSC-A vs SSC-A (left)), myeloid (HLA-DR+ Lin1- CD11c + (middle)) or plasmacytoid DCs (HLA-DR+ Lin1- CD123+ (right)). d Delta of concentration in pg/mL of IL-12p70 secreted in culture supernatants (difference in concentration secreted by mature and immature) DCs from HD (white box, n = 10) and patients before (gray box, n = 17) and after chemotherapy (dashed box, n = 17). Boxes and whiskers 10–90%, two-way ANOVA analysis, with Turkey’s multiple comparison tests, * p < 0.05, ** p < 0.01

Fig. 3

Suppressed T cell responsiveness in BC patients before chemotherapy. a Representative contour plots of T cells (SSC-A vs. CD3+) of HD unstimulated or stimulated 24 h with anti-CD3/CD28/CD2 beads (numbers represent MFI of CD3). b Quantification of CD3 MFI in PBMCs of HD in response to in vitro stimulation (left panel), and delta of CD3 MFI from HD (white box), and BC patients before (gray box) and after chemotherapy (dashed box – right panel). c Representative contour plots of T cell (gated on CD3+) activation phenotype (CD154 vs. CD69) of cells obtained from HD in response to in vitro stimulation with anti-CD3/CD28/CD2 beads, numbers represent the percentage of each population. d Quantification of MFI of each activation marker (delta of stimulated minus unstimulated cells) of CD25 (left panel), CD69 (middle panel) and CD154 (right panel) of HD (white box), and BC patients before (gray box) and after chemotherapy (dashed box). Box and whiskers 10–90%. HD (n = 12), patients before (n = 17) and after chemotherapy (n = 17). Non-parametric t-test (panel B – left panel) and Two-way ANOVA analysis, with Turkey’s multiple comparison tests, * p < 0.05, ** p < 0.01

Fig. 4

The predictive capacity of immune readouts for clinical response to chemotherapy. a Scatter plot of percentage of CD3 internalization vs. residual cancer burden (RCB) index in BC patients after chemotherapy (Pearson correlation = −0.583, p < 0.05). b Predictive value of TCR (CD3) internalization (left panel) and the delta percentage of CD83 expression in plasmacytoid DCs evaluated before therapy and compared in patients with or without better clinical response (pCR/RCB-I vs RCB-II respectively). c ROC curves of TCR internalization (AUC = 0.816, p = 0.0452), and delta percentage of CD83 expression in plasmacytoid DCs to predict tumor response (AUC = 0.825, p = 0.039). Box and whiskers 10–90%, Pearson correlation test, Mann-Whitney test, * p < 0.05, ** p < 0.01