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. 2017 Feb 6;8:87.
doi: 10.3389/fimmu.2017.00087. eCollection 2017.

In Vivo Efficacy of Umbilical Cord Blood Stem Cell-Derived NK Cells in the Treatment of Metastatic Colorectal Cancer

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Free PMC article

In Vivo Efficacy of Umbilical Cord Blood Stem Cell-Derived NK Cells in the Treatment of Metastatic Colorectal Cancer

John P Veluchamy et al. Front Immunol. .
Free PMC article

Abstract

Therapeutic monoclonal antibodies against the epidermal growth factor receptor (EGFR) act by inhibiting EGFR downstream signaling and by eliciting a natural killer (NK) cell-mediated antitumor response. The IgG1 mAb cetuximab has been used for treatment of RASwt metastatic colorectal cancer (mCRC) patients, showing limited efficacy. In the present study, we address the potential of adoptive NK cell therapy to overcome these limitations investigating two allogeneic NK cell products, i.e., allogeneic activated peripheral blood NK cells (A-PBNK) and umbilical cord blood stem cell-derived NK cells (UCB-NK). While cetuximab monotherapy was not effective against EGFR- RASwt, EGFR+ RASmut, and EGFR+ BRAFmut cells, A-PBNK were able to initiate lysis of EGFR+ colon cancer cells irrespective of RAS or BRAF status. Cytotoxic effects of A-PBNK (but not UCB-NK) were further potentiated significantly by coating EGFR+ colon cancer cells with cetuximab. Of note, a significantly higher cytotoxicity was induced by UCB-NK in EGFR-RASwt (42 ± 8 versus 67 ± 7%), EGFR+ RASmut (20 ± 2 versus 37 ± 6%), and EGFR+ BRAFmut (23 ± 3 versus 43 ± 7%) colon cancer cells compared to A-PBNK and equaled the cytotoxic efficacy of the combination of A-PBNK and cetuximab. The antitumor efficacy of UCB-NK cells against cetuximab-resistant human EGFR+ RASmut colon cancer cells was further confirmed in an in vivo preclinical mouse model where UCB-NK showed enhanced antitumor cytotoxicity against colon cancer independent of EGFR and RAS status. As UCB-NK have been proven safe in a recently conducted phase I clinical trial in acute myeloid leukemia, a fast translation into clinical proof of concept for mCRC could be considered.

Keywords: A-PBNK; EGFR; RAS mutation; UCB-NK; allogeneic NK cell immunotherapy; cetuximab; metastatic colorectal cancer.

Figures

Figure 1
Figure 1
Low prevalence and functionally impaired natural killer (NK) cells in colorectal cancer (CRC) patients. (A) Frequency of NK cells within peripheral blood mononuclear cells from healthy controls and from metastatic CRC (mCRC) patients at baseline and after the first cycle of chemotherapy. (B) NK cell degranulation in healthy controls and mCRC patients after a 4-h coculture of resting NK cells with A431 cells in the presence (open symbols) or absence (closed symbols) of cetuximab at an E:T ratio of 1:1. (C) Expression levels of resting NK cell CD16 and (D) NKp44 in healthy controls and in mCRC patients before and after one cycle of chemotherapy. Data represent mean ± SEM from 10 mCRC patients and 10 age- and sex-matched healthy controls. *P < 0.05, **P < 0.01, ***P < 0.005, calculated with one-way ANOVA, multiple comparison between column means.
Figure 2
Figure 2
Ex vivo cytotoxic efficacy of A-PBNK and UCB-NK cells against colorectal cancer (CRC) cells. CRC cell lines COLO320 (EGFR, RASwt), SW480 (EGFR+, RASmut), and HT-29 (EGFR+, RASwt, BRAFmut) were subjected to natural killer (NK) killing using two allogeneic NK cell products, i.e., A-PBNK and UCB-NK cells. 7-AAD (A,B,C) and CD107a (D,E,F) were measured after a 4-h coculture of A-PBNK and UCB-NK cells with CRC targets in the presence or absence of cetuximab at an E:T ratio of 1:1. Experiments were carried out in triplicate. Bars represent mean ± SEM, n = 5. *P < 0.05 and **P < 0.01, calculated with two-way ANOVA, multiple comparison between column means.
Figure 3
Figure 3
Significant antitumor effects of UCB-NK cells in vivo. Real-time monitoring of tumor progression and treatment response was performed measuring Gluc levels from mice blood twice a week. Baseline Gluc values were obtained from all mice a day before tumor injection (day 1), and further monitoring continued until day 35. Blood Gluc levels were compared between control SW480 only (A) group and treatment groups SW480 + cetuximab (B), SW480 + UCB-NK (C), and SW480 + UCB-NK + cetuximab (D) for statistical significance. Data presented is from six mice per group (n = 6). Scatter plots represent mean ± SEM. *P < 0.05, calculated with unpaired t test.
Figure 4
Figure 4
Successful tumor elimination by UCB-NK cells as revealed by bioluminescence imaging in vivo. (A) Four mice from control and treatment groups were imaged at day 35 for tumor load and distribution. Mice were injected retro-orbitally with Gluc substrate coelenterazine and images were acquired for 5 min. In SW480 control and SW480 + cetuximab groups, tumor growth was extensive and highly disseminated, spreading to most parts of the body. However, in UCB-NK and UCB-NK + cetuximab groups, there was a significantly lower tumor load, which was further verified by calculating the average radiance between groups as shown in panel (B) (n = 4 mice per group). (C) Cetuximab functionality against EGFR+++ RASwt A431 cells was tested in parallel to SW480 studies in BRGS mice (n = 3 mice per group). For panels (B,C), bars represent mean ± SEM. *P < 0.05 for panels (B,C) was calculated with unpaired t test.
Figure 5
Figure 5
Significant survival benefit in cetuximab-resistant RAS mutant tumor bearing mice treated with UCB-NK cells. Kaplan–Meier survival curves were plotted for the total experimental study period from day 0 until day 65. Survival rates of SW480 (EGFR+, RASmut) tumor-bearing mice (n = 6 per group) following treatment with PBS only (black line), cetuximab only (blue line), UCB-NK only (green line), and UCB-NK + cetuximab (orange line) were plotted over time to monitor treatment outcome. Statistical differences between groups were calculated using log-rank (Mantel–Cox) test and indicated in the figure.

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