Increased circulating myeloid-derived suppressor cells correlate with clinical cancer stage, metastatic tumor burden, and doxorubicin-cyclophosphamide chemotherapy

Abstract

Abnormal accumulation of myeloid-derived suppressor cells (MDSC) is an important mechanism of tumor immune evasion. Cyclophosphamide (CTX) has also been shown in non-tumor bearing animals to cause transient surges in MDSC. Knowledge of MDSC is primarily based on preclinical work, and to date only few published studies have involved cancer patients. The goal of this study was to test the hypothesis that circulating MDSC levels correlate with clinical cancer stage, CTX-based chemotherapy, and metastatic tumor burden. Whole blood was collected from 106 newly diagnosed solid tumor patients (stages I-IV). Percentages of circulating MDSC (Lin(-/Lo), HLA DR-, CD33(+)CD11b(+)) were determined prior to initiation of systemic therapy. In 17 early stage breast cancer patients receiving doxorubicin-cyclophosphamide chemotherapy every 14 days (ddAC) blood was collected on day 1 of each cycle. Circulating MDSC were significantly increased in cancer patients of all stages relative to healthy volunteers. A significant correlation between circulating MDSC and clinical cancer stage was also observed. Moreover, among stage IV patients, those with extensive metastatic tumor burden had the highest percent and absolute number of MDSC. Significant increases in circulating MDSC were observed with ddAC when compared with pretreatment levels. Circulating MDSC levels correlate with clinical cancer stage, ddAC, and metastatic tumor burden. This information must be incorporated into the design of future trials exploring immune-based therapeutic strategies. Pharmacologic modulation of MDSC should also be tested in future clinical trials.

Fig. 1

Immunophenotyping of MDSC by flow cytometry. To determine the percentage of MDSC in patients, fresh whole blood was incubated with a mixture of anti-Lin, HLA DR, CD33 and CD11b mAbs. Acquired cells were first gated (R1) based on the expression of Lin and HLA DR. R1 was comprised of Lin^−/Lo and HLA DR⁻ cells. Within this population the fraction of cells expressing both CD33 and CD11b was determined. Therefore, MDSC were defined as Lin^−/Lo, HLA DR⁻, CD33⁺ and CD11b⁺ cells. MDSC percentage was calculated as percentage of total nucleated cells in whole blood samples. Representative flow diagrams of a a healthy volunteer, b a patient with stage IV breast cancer, and c a breast cancer patient receiving ddAC chemotherapy

Fig. 2

Absolute number and total percentage of circulating MDSC correlates with clinical tumor stage. Whole blood drawn from patients prior to any therapy was analyzed for the presence of MDSC. Results showed a significantly higher percentage of circulating MDSC in cancer patients (n = 106) versus normal volunteers (n = 21) (2.85 vs. 1.26%; P < 0.0001) (a). Moreover, both greater percentages (b) and absolute numbers (MDSC/μL) (c) of MDSC were detected in cancer patients with stage IV disease relative to patients with clinical stages I/II and stage III solid tumors. Mean estimates for each stage and 95% confidence intervals for the mean are shown to the right of the data. Differences between normal volunteers (1.26%) and cancer patients of all stages (I/II 1.96%; III 2.46%; IV 3.77%) were statistically significant (P < 0.01). Likewise, differences in percent and absolute number of MDSC between stage IV cancer patients and stages I/II and III were statistically significant (P < 0.03). Differences in percentage and absolute MDSC between stages I/II and stage III cancer patients were not statistically significant (P = 0.22 and 0.23, respectively). However, the increasing pattern with stage and narrow intervals indicate strong evidence of an increasing trend (trendtest P < 0.001)

Fig. 3

Circulating MDSC levels correlate with extensive metastatic tumor burden in patients with advanced stage IV solid tumors. Patients with advanced clinical stage were divided into limited (n = 20) and extensive (n = 36) tumor burden. Patients with extensive metastatic tumor burden must have had either: diffuse involvement of one organ system or ≥3 or more distinct organ sites involved. Patients with extensive tumor burden had significantly higher percentages (a) than patients with limited metastatic tumor burden with 4.37 and 2.9%, respectively (P = 0.017) and (b) absolute numbers of circulating MDSC (325.7 vs. 177.94 μL⁻¹, respectively; P ≤ 0.01). Mean estimates and 95% confidence intervals for each group are shown to the right of the data

Fig. 4

Stage IV cancer patients with limited or extensive tumor burden and correlation of MDSC levels with disease progression. a PET/CT scan of a patient with stage IV adrenocortical carcinoma with a large 10 × 12 cm adrenal mass (left arrow) and extensive bony metastases (not shown); a patient with melanoma of the left sinus (right arrow) measuring 15 × 12 cm, and bony, liver, and brain metastases; a patient with prior stage II breast cancer with recurrence in left axillary (white arrowhead) and left supraclavicular nodes (not shown). The corresponding % MDSC is shown above. b Percentages of circulating MDSC in six patients with stage IV cancer drawn prior to initiation of systemic therapy and again at each time when CT scans were obtained to determine radiographic response. Radiographic responses (black arrows) were associated with corresponding decreases in circulating MDSC levels. Likewise, MDSC levels were found to increase at the time of disease progression (gray arrows)

Fig. 5

ddAC, but not ddT, was associated with significant increases in circulating MDSC in early stage breast cancer patients (n = 19). Data from day 1 of all four cycles of ddAC for each individual were averaged, and also for day 1 of all four cycles of ddT. A regression model, accounting for correlation with the patient was applied. Means for individual patients at each treatment phase (BL, ddAC, or ddT) are shown using small circles and observations from within the same patient are connected using thin lines. Based on the regression model, mean estimates at each treatment phase are shown using black circles and connected via a thick line. 95% confidence intervals (estimated by regression model) are shown for each treatment phase. Results showed significantly greater percentages (a) and absolute numbers (b) of MDSC with ddAC compared to both BL and during paclitaxel. All differences were statistically significant (P < 0.01). c Schematic representation of ddAC and ddT chemotherapy with growth factor support received by all 19 breast cancer patients

Fig. 6

Increased levels of circulating MDSC correlate with decreased T cell responses. Equal numbers of PBMC from two normal volunteers (N), two patients with advanced disease (P10 and P59), and a breast cancer patient (P26) before (BL) and 14 days after one course of ddAC chemotherapy were assayed for cell proliferation, IL-2 and IFN-γ secretion in response to activation with anti-CD3/CD28-coated beads. Cell proliferation and cytokine release was determined 3 days after activation. Corresponding percentages of circulating MDSC are shown above in bold

Fig. 7

Direct contact of T cells with isolated myeloid cells (CD33⁺) from cancer patients inhibits T cell activation. Myeloid cells (CD33⁺) were isolated from freshly drawn blood from two normal volunteers (N1 and N2), two patients with stage IV cancer (P37 and P38), and a breast cancer patient receiving ddAC chemotherapy (BR1). a Percentage of circulating MDSC at the time of CD33⁺ isolation. b Representative histograms of CD33⁺ fractions before and after enrichment. c Proliferation of isolated autologous T cells in response to CD3/CD28 activation in the presence of the indicated ratios of purified autologous CD33⁺ cells. Results are the average of triplicate measurements ± SDEV