Genetic engineering of human NK cells to express CXCR2 improves migration to renal cell carcinoma

Abstract

Background: Adoptive natural killer (NK) cell transfer is being increasingly used as cancer treatment. However, clinical responses have so far been limited to patients with hematological malignancies. A potential limiting factor in patients with solid tumors is defective homing of the infused NK cells to the tumor site. Chemokines regulate the migration of leukocytes expressing corresponding chemokine receptors. Various solid tumors, including renal cell carcinoma (RCC), readily secrete ligands for the chemokine receptor CXCR2. We hypothesize that infusion of NK cells expressing high levels of the CXCR2 chemokine receptor will result in increased influx of the transferred NK cells into tumors, and improved clinical outcome in patients with cancer.

Methods: Blood and tumor biopsies from 14 primary RCC patients were assessed by flow cytometry and chemokine analysis. Primary NK cells were transduced with human CXCR2 using a retroviral system. CXCR2 receptor functionality was determined by Calcium flux and NK cell migration was evaluated in transwell assays.

Results: We detected higher concentrations of CXCR2 ligands in tumors compared with plasma of RCC patients. In addition, CXCL5 levels correlated with the intratumoral infiltration of CXCR2-positive NK cells. However, tumor-infiltrating NK cells from RCC patients expressed lower CXCR2 compared with peripheral blood NK cells. Moreover, healthy donor NK cells rapidly lost their CXCR2 expression upon in vitro culture and expansion. Genetic modification of human primary NK cells to re-express CXCR2 improved their ability to specifically migrate along a chemokine gradient of recombinant CXCR2 ligands or RCC tumor supernatants compared with controls. The enhanced trafficking resulted in increased killing of target cells. In addition, while their functionality remained unchanged compared with control NK cells, CXCR2-transduced NK cells obtained increased adhesion properties and formed more conjugates with target cells.

Conclusions: To increase the success of NK cell-based therapies of solid tumors, it is of great importance to promote their homing to the tumor site. In this study, we show that stable engineering of human primary NK cells to express a chemokine receptor thereby enhancing their migration is a promising strategy to improve anti-tumor responses following adoptive transfer of NK cells.

Keywords: Adoptive cell therapy; CXCR2; Chemokines; NK cells; Renal cell carcinoma.

Fig. 1

Expression of CXCR2 on NK cells and its ligands on RCC tumors. a Expression of CXCR2 ligands in the plasma and tumor lysate of patients with primary RCC relative to mg total protein (n = 14). Samples were analyzed using Bio-Plex Pro Human Chemokine 40-plex panel. b Expression of CXCR2 ligands by primary low-passage (P1 or P3) RCC cell lines. CXCL1 production by TINCA3 and TINCA7 as well as CXCL8 production by TINCA3 were above the quantification limits of 13,990 pg/mL and 31,093 pg/mL, respectively. c Pearson correlation of CXCL5 levels in tumor lysate in patients with primary RCC and frequency of intratumoral CXCR2-positive NK cells (n = 9). d Frequency and (e) levels of CXCR2 expression by NK cells in peripheral blood (PB) and primary tumors of RCC patients (n = 13). A representative histogram from patient RCC007 is shown. f Flow cytometry analysis of CXCR2 expression by healthy donor peripheral blood non-activated NK cells and eight-day expanded NK cells. Results are representative of four experiments

Fig. 2

Retroviral transduction and functionality of NK cells. a Top and middle: Schematic representation of retroviral vectors containing human CXCR2 and NGFR, respectively, and representative histograms of the transgene expression on non-transduced (NT), CXCR2- and NGFR-transduced primary NK cells. Bottom: Flow cytometry analysis of CXCR2 and NGFR expression on NK cells after the transduction (n = 24). Horizontal bars represent the mean transduction efficiency. b NK cell-mediated cytotoxicity against K562 cells after 5 h. One of four representative experiments is shown. NK cell-mediated cytotoxicity against (c) ACHN and (d) Caki-2 cells after 20 h. One of five and three representative experiments is shown, respectively. e Flow cytometry analysis of NK cell degranulation after stimulation with K562 at a E:T ratio of 1:1 (n = 4) and with ACHN and Caki-2 at a E:T ratio of 2:1 to 1:1 (n = 3 for each cell line). f Flow cytometry analysis of NK cell IFN-γ production after stimulation with K562. Data in e and f are depicted as mean ± SEM of fold-change compared with non-transduced NK cells and analyzed with repeated measures one-way ANOVA. g Proliferation of non-transduced (NT), NGFR-transduced, and CXCR2-transduced NK cells after 7 days as assessed by CFSE staining. Results are representative of three experiments

Fig. 3

Adhesion of NK cells incorporating the transgenes. a Flow cytometry analysis of CD11a (n = 6) and CD11b (n = 4) expression on NK cells incorporating (CXCR2+ and NGFR+) and not incorporating the respective transgene (CXCR2- and NGFR-). b Representative dot plots depicting counts of collected events within the double-positive gate after 0-min and 10-min co-cultures of CFSE-labeled K562 cells and BMQC-labeled NK cells. c NK cells incorporating and not incorporating the CXCR2 (n = 5) or NGFR (n = 6) transgenes in conjugates with K562 cells after 10 min of co-culture. d Degranulation against K562 of CXCR2- and CXCR2+ NK cells, as assessed by flow cytometry (n = 4)

Fig. 4

Calcium mobilization in transduced NK cells. a Multiplex analysis of CXCR2 ligands in the supernatants of the RCC cell lines ACHN, 786-O, MAR, CAKI-2, and A498. CXCL1 in ACHN cells was above the quantification limit of 13,990 pg/mL, CXCL5 in A498 cells was below the quantification limit of 603 pg/mL. b Calcium mobilization in CXCR2- and NGFR-transduced NK cells stimulated with recombinant CXCL8 (50 ng/mL) or supernatant from the RCC cell lines ACHN, MAR or A498. Values are Fluo-3 relative fluorescent units (RFU) normalized to the baseline prior to the addition of stimuli. c Calcium response in CXCR2- and NGFR-transduced NK cells after addition of the stimuli calculated as the normalized area under curve (AUC)

Fig. 5

CXCR2-specific migration of CXCR2-transduced NK cells. a Time course of the migration of non-transduced (NT), NGFR- or CXCR2-transduced NK cells toward a pool of recombinant CXCL1, CXCL2, CXCL3, and CXCL8. Cell migration was analyzed with the IncuCyte ZOOM live cell imager and expressed as area occupied by NK cells on the top surface normalized to the initial top value (n = 4 per condition). Repeated measures two-way ANOVA was applied to analyze data. b Transwell migration assay of NT, NGFR- or CXCR2-transduced NK cells toward a pool of the recombinant CXCR2 ligands CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8. The number of migrated cells was determined by automatic counting using a flow cytometer from three technical replicates per experiment (n = 7). c NK cell-mediated cytotoxicity against K562 cells after transwell migration toward medium with or without recombinant CXCR2 ligands (n = 3). d Equal concentrations of CXCR2 ligands were added to the upper and lower chambers of a transwell assay of NGFR- and CXCR2-transduced NK cells. Results are representative of three experiments with two technical replicates. e Transwell assay of NGFR- and CXCR2-transduced NK cells toward recombinant CXCL1, CXCL8 and CXCL5 following pre-incubation with the selective CXCR2 inhibitor SB225002. Results are representative of three experiments for CXCL1 and CXCL8 and of two experiments for CXCL5 with two technical replicates. f Transwell assay of NGFR- and CXCR2-transduced NK cells toward supernatants from RCC cell lines. Data are mean values from three technical replicates per experiment (n = 4 for ACHN, 786-O, and MAR; n = 3 for Caki-2 and A498). Percent migrated cells are calculated based on total cell input. *, P < 0.05, **, P < 0.01 and ****, P < 0.0001