Metronomic capecitabine as an immune modulator in glioblastoma patients reduces myeloid-derived suppressor cells

Abstract

BACKGROUNDMyeloid-derived suppressor cells (MDSCs) are elevated in the circulation of patients with glioblastoma (GBM), present in tumor tissue, and associated with poor prognosis. While low-dose chemotherapy reduces MDSCs in preclinical models, the use of this strategy to reduce MDSCs in GBM patients has yet to be evaluated.METHODSA phase 0/I dose-escalation clinical trial was conducted in patients with recurrent GBM treated 5-7 days before surgery with low-dose chemotherapy via capecitabine, followed by concomitant low-dose capecitabine and bevacizumab. Clinical outcomes, including progression-free and overall survival, were measured, along with safety and toxicity profiles. Over the treatment time course, circulating MDSC levels were measured by multiparameter flow cytometry, and tumor tissue immune profiles were assessed via time-of-flight mass cytometry.RESULTSEleven patients total were enrolled across escalating dose cohorts of 150, 300, and 450 mg bid. No serious adverse events related to the drug combination were observed. Compared with pretreatment baseline, circulating MDSCs were found to be higher after surgery in the 150-mg treatment arm and lower in the 300-mg and 450-mg treatment arms. Increased cytotoxic immune infiltration was observed after low-dose capecitabine compared with untreated GBM patients in the 300-mg and 450-mg treatment arms.CONCLUSIONSLow-dose, metronomic capecitabine in combination with bevacizumab was well tolerated in GBM patients and was associated with a reduction in circulating MDSC levels and an increase in cytotoxic immune infiltration into the tumor microenvironment.TRIAL REGISTRATIONClinicalTrials.gov NCT02669173.FUNDINGThis research was funded by the Cleveland Clinic, Case Comprehensive Cancer Center, the Musella Foundation, B*CURED, the NIH, the National Cancer Institute, the Sontag Foundation, Blast GBM, the James B. Pendleton Charitable Trust, and the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation. Capecitabine was provided in kind by Mylan Pharmaceuticals.

Keywords: Cancer immunotherapy; Clinical Trials; Immunotherapy; Oncology.

Figure 1. Study schematic demonstrating the time points for capecitabine treatment and immune analysis via flow cytometry and CyTOF.

Figure 2. Peripheral MDSCs are reduced over time in patients treated with capecitabine at 300 mg bid and 450 mg bid.

Flow cytometry analysis of PBMCs longitudinally identified as MDSCs (HLA-DR^–/lo, CD11b⁺, CD33⁺), and the log fold change in MDSCs per patient after surgical resection is depicted (A), with each symbol representing the blood draws in sequential order from 1 to 13 (n = 10 reference cohort, n = 4 at 150 mg bid, n = 3 at 300 mg bid, n = 4 at 450 mg bid). The average log fold change of MDSCs per patient over time is graphed per treatment group (B) and identified a significant difference between untreated and all treatment groups and a maximal reduction in the 300-mg bid (n = 3) and 450-mg bid treatment groups (n = 3) (B). Error bars represent SDs. One-way ANOVA was used to analyze these data; the F test, with and without the reference untreated cohort included as a group, yielded P < 0.0001 and P = 0.0308 respectively.

Figure 3. Capecitabine increases the immune activation in tumors after 7 days of treatment before surgery.

CyTOF analysis using an immune panel of 28 immune markers analyzed capecitabine-treated tumor samples from patients 5, 6, 9, 10, and 11, along with newly diagnosed tumor samples (patients 1, 2, 3), and 1 recurrent GBM tumor (sample 1, n = 4 total) (A) is represented as a tSNE multidimensional plot and colored by CD45 expression, highlighting the immune populations. After selecting immune populations based on CD45 expression, all tumor sample immune cells were combined and used to cluster immune populations in an unbiased manner from live/CD45⁺ cells only (B). Separate newly diagnosed GBM patient (n = 3), recurrent GBM patient (n = 1), 300-mg bid capecitabine-treated patient (n = 2), and 450-mg bid capecitabine-treated patient (n = 3) tSNE plots represent the immune landscape of each tumor cohort (C).

Figure 4. Comparison of untreated versus capecitabine-treated immune populations on a per-patient basis.

Unbiased clustering identification of immune populations and quantification of the proportion of each cell type present in the CD45⁺ population are represented as a proportion of the total live/CD45⁺ (n = 1 patient per bar). Statistical analysis comparing untreated versus treated immune populations identified statistically significant differences between the populations. Linear models of the data with 2-tailed t test comparisons and Benjamini-Hochberg were used to control for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001. Graphs represent data sets as median with first and third quartiles.

Figure 5. Using patient tumor CyTOF data, a machine-learning approach identified a reduction in a signature for immune cell exhaustion in the tumors of capecitabine-treated patients.

From the CyTOF data, a decision tree was generated using the CytoDx R package (A). The first node of the decision tree is highlighted, identifying the initial finding of 76% of patients with a lower level of CTLA-4⁺ cells. Multidimensional tSNE modeling of the total CD45⁺ cells from the tumors of untreated and treated patients, colored by CTLA-4 expression levels, identifies the clusters with a reduction in CTLA-4 upon treatment (B). Manual gating of the CyTOF data highlighted the quantitative differences in CTLA-4⁺ lymphocytes in the tumors of patients treated with capecitabine (C). Further manual gating for the final subset of CTLA-4^– cells identified by the decision tree revealed a unique population of CTLA-4⁺PD-1⁺ macrophages that were suppressed upon capecitabine treatment (D) (untreated n = 4, capecitabine treated n = 5).