Intraperitoneal injection of in vitro expanded Vγ9Vδ2 T cells together with zoledronate for the treatment of malignant ascites due to gastric cancer

Abstract

Malignant ascites caused by peritoneal dissemination of gastric cancer is chemotherapy-resistant and associated with poor prognosis. We conducted a pilot study to evaluate the safety of weekly intraperitoneal injections of in vitro expanded Vγ9Vδ2 T cells together with zoledronate for the treatment of such malignant ascites. Patient peripheral blood mononuclear cells were stimulated with zoledronate (5 μmol/L) and interleukin-2 (1000 IU/mL). After 14 days culture, Vγ9Vδ2 T-cells were harvested and administered intraperitoneally in four weekly infusions. The day before T-cell injection, patients received zoledronate (1 mg) to sensitize their tumor cells to Vγ9Vδ2 T-cell recognition. Seven patients were enrolled in this study. The number of Vγ9Vδ2 T-cells in each injection ranged from 0.6 to 69.8 × 10(8) (median 59.0 × 10(8)). There were no severe adverse events related to the therapy. Intraperitoneal injection of Vγ9Vδ2 T cells allows them access to the tumor cells in the peritoneal cavity. The number of tumor cells in the ascites was significantly reduced even after the first round of therapy and remained substantially lower over the course of treatment. IFN-γ was detected in the ascites on treatment. Computed tomography revealed a significant reduction in volume of ascites in two of seven patients. Thus, injection of these antitumor Vγ9Vδ2 T-cells can result in local control of malignant ascites in patients for whom no standard therapy apart from paracentesis is available. Adoptively transferred Vγ9Vδ2 T-cells do indeed recognize tumor cells and exert antitumor effector activity in vivo, when they access to the tumor cells.

Keywords: Gastric cancer; Vγ9Vδ2 T-cell; malignant ascites; peritoneal dissemination; zoledronate.

© 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Figures

Figure 1

(A) Intraperitoneal administration of zoledronate sensitizes tumor cells to Vγ9Vδ2 T-cell recognition. Isopentenyl pyrophosphate (IPP) is an intermediate metabolite in the mevalonate–cholesterol pathway, recognized by Vγ9Vδ2 T-cells. Zoledronate inhibits farnesyl pyrophosphate (FPP) synthase, thereby causing the accumulation of IPP and triphosphoric acid I-adenosine-50-yl ester 3-(3-methylbut-3-enyl) ester (ApppI) in the tumor cells. When Vγ9Vδ2 T-cells are injected into the peritoneal cavity, they can recognize IPP and respond to tumor cells. (B) The study scheme of weekly i.p. Vγ9Vδ2 T-cell injection. In the first series of injections, zoledronate (1 mg) was administered i.v. on day 0, followed by i.p. Vγ9Vδ2 T-cell injection on day 1. Zoledronate was i.p. injected via a catheter from the second to fourth injection.

Figure 2

IPP accumulation by zoledronate was evaluated by the CD107 translocation assay of Vγ9Vδ2 T-cells. Daudi cells were preincubated overnight with indicated concentration of zoledronate (0, 1, 5, 10, 50, and 100 μmol/L) or 10 μmol/L pravastatin sodium and used as stimulator cells. The 5 × 105 Daudi cells were incubated with the same number of Vγ9Vδ2 T-cells for 2 h at 37°C in the presence of GolgiStop and anti-CD107a/b mAbs. Vγ9Vδ2 T-cells were also stimulated with PMA (20 ng/mL)/ionomycin (2 μg/mL). CD107 translocation was measured by flow cytometry. Results were expressed as percentages of positive cells within the Vγ9Vδ2 T-cell population.

Figure 3

Dynamics of Vγ9Vδ2 T cells and responses in patients with malignant ascites. (A) The number of Vγ9Vδ2 T cells in ascites. The ascites fluid was drained from the peritoneal cavity via the indwelling catheter before zoledronate and Vγ9Vδ2 T-cell injections and 24 h after Vγ9Vδ2 T-cell injections. The cells were isolated by density gradient centrifugation and stained with anti-CD45, -CD3, and -TCRVγ9. The stained cells were analyzed on flow cytometry and the numbers of Vγ9Vδ2 T cells calculated. (B) IFN-γ concentration (pg/mL) in ascites at the indicated time points was measured by the FlowCytomix bead assay. (C) The cells from ascites were also stained with anti-EpCAM mAb and the numbers of EpCAM+ tumor cells calculated. *Sample was collected 4 h after i.p. Vγ9Vδ2 T-cell injection.

Figure 4

The cellular components and appearance of the ascites fluid. (A) The ascites fluid was harvested 24 h after Vγ9Vδ2 T-cell injection; the cells were stained with anti-TCRVγ9-FITC and anti-EpCAM-APC mAbs and examined by confocal fluorescence microscopy. The EpCAM+ tumor cells (blue) are attached to and surrounded by Vγ9Vδ2 T cells (green) in ascites after Vγ9Vδ2 T-cell injections. Magnification was 150× on the left and 600× on the right. (B) Smears were prepared, air-dried, and stained with Diff-Quik (Sysmex, Kobe, Japan) according to the manufacturer's instructions. Cell morphology was evaluated using bright field microscopy (OLYMPUS BX41 with Canon EOS Kiss X4 digital camera, OLYMPUS, Tokyo, Japan, magnification 200×). Data from patient 2328 on day 0 (a: before zoledronate i.v.), day 9 (b: 24 h after 2nd Vγ9Vδ2 T-cell injection), day 21 (c: before zoledronate i.p.), and day 22 (d: 4 h after Vγ9Vδ2 T-cell injection) are shown. (C) The appearance of ascites from patient 2325 before and after four courses of Vγ9Vδ2 T-cell injections. (D) γδ T cells from patient 2325 (green staining with CFSE) recognized and killed autologous EpCAM+ gastric cancer cells purified from ascites fluid (red staining with PKH-26), by direct contact. Tumor cells were attacked by the γδ T cells; collapse of the cell membranes led to apoptosis. It took approximately 2 h to progress from (a) to (c). Movie clip S1 is also provided.

Figure 5

Computed tomography findings in patients 2325 (A) and 2328 (B). Retention of a large amount of ascites was observed before treatment (left panels). The amount of ascites was reduced 1 week (middle panels) and 4 weeks (right panels) after four courses of Vγ9Vδ2 T-cell injections.