Activated human primary NK cells efficiently kill colorectal cancer cells in 3D spheroid cultures irrespectively of the level of PD-L1 expression

Abstract

Haploidentical Natural Killer (NK) cells have been shown as an effective and safe alternative for the treatment of haematological malignancies with poor prognosis for which traditional therapies are ineffective. In contrast to haematological cancer cells, that mainly grow as single suspension cells, solid carcinomas are characterised by a tridimensional (3D) architecture that provide specific surviving advantages and resistance against chemo- and radiotherapy. However, little is known about the impact of 3D growth on solid cancer immunotherapy especially adoptive NK cell transfer. We have recently developed a protocol to activate ex vivo human primary NK cells using B lymphoblastic cell lines, which generates NK cells able to overcome chemoresistance in haematological cancer cells. Here we have analysed the activity of these allogeneic NK cells against colorectal (CRC) human cell lines growing in 3D spheroid culture and correlated with the expression of some of the main ligands regulating NK cell activity. Our results indicate that activated NK cells efficiently kill colorectal tumour cell spheroids in both 2D and 3D cultures. Notably, although 3D CRC cell cultures favoured the expression of the inhibitory immune checkpoint PD-L1, it did not correlate with increased resistance to NK cells. Finally, we have analysed in detail the infiltration of NK cells in 3D spheroids by microscopy and found that at low NK cell density, cell death is not observed although NK cells are able to infiltrate into the spheroid. In contrast, higher densities promote tumoural cell death before infiltration can be detected. These findings show that highly dense activated human primary NK cells efficiently kill colorectal carcinoma cells growing in 3D cultures independently of PD-L1 expression and suggest that the use of allogeneic activated NK cells could be beneficial for the treatment of colorectal carcinoma.

Keywords: 3D Spheroids cultures; Colorectal Carcinoma; Immunotherapy; Natural Killer Cells Activation; allogeneic human primary Natural Killer Cells.

Figure 1.

Spheroid characterization. (A) Dot-Plot of a representative spheroid control showing the percentage of viable cells after 4 days (Ann-V⁻/AAD⁻) for the three cell lines. PS exposure on plasma membrane (annexin-V) and membrane permeabilization (7AAD) were analysed by flow cytometry as described in Material and Methods. (B) CRC spheroid area evolution in control group shows tumour progression in absence of treatment along the study time course. (C) Monocellular suspensions from 2D or 3D cultures were analysed by flow cytometry for the expression of HLA-ABC, ICAM-1 and PD-L1. Bar charts represent the Mean Fluorescence Intensity in 2D (white) and 3D (black) conditions. Histograms show a representative determination for HCT116 cell line. The dotted line corresponds to isotype control, the black line to 2D conditions and the grey line to 3D conditions. Data in the graphics are represented as the mean±SEM of at least 5 independent experiments where duplicate measures were determined as described in Materials and Methods. n.s. no statistically significance or *p<0.05, **p<0.01, ***p<0.001 statistically significance was analysed by t-test.

Figure 2.

Apoptosis induced by R69-LCL activated NK cells on 2D and 3D HCT116 cell cultures. NK cells were enriched by MACS from fresh PBMC or after activation for 5 days with R69-LCLs (aNK). Subsequently they were incubated with Caco-2, HT29 and HCT116 cells seeded in monolayers for 4 hours (A) or spheroids for 48.hours (B) at low (up to 3:1) or high (between 6:1 and 9:1) e:t ratios. PS exposure on plasma membrane (annexin-V) and membrane permeabilization (7AAD) were analysed by flow cytometry as described in Materials and Methods. Results are presented as mean +/− SEM SEM of at least 3 independent donors

Figure 3.

Cytotoxic effect of NK R69-LCL activated cells on HCT116 spheroid compared with naïve NK cells at high e:t ratio. (A, B and C) Fluorescent images of HCT116 cells (green) and NK cells (red) in a high e:t ratio during 96 hours treatment. Area evolution of CRC spheroids after treatment with nNK (A), activated NK cells (B), and control group with no NK presence (C). (D,E) A representative experiment from at least three experiments performed with 3 different donors is shown. The relative spheroid area was measured (D) Naïve nNK cells were able to reduce partially the tumour size but after 72h, spheroids star to grow and no clear effect is observed after 96h. (E) Activated NK cells at high e:t ratio are able to diminish drastically CRC spheroid size until almost complete elimination of GFP signal expressed by CRC cells.

Figure 4.

The e:t Ratio determines the cytotoxic effect of activated NK cells. A representative experiment from at least three experiments performed with 3 different donors is shown (A, B, C and D) Fluorescent images of HCT116 cells (green) and NK cells (red) in different e:t ratio after 96 hours of co-culture. (E, F and G) Relative area progression of the spheroids after treatment with different e:t ratio of activated NK cells from 3 different donors. (B and E) correspond to a 3:1 ratio of activated NK cells, (C and F) shows results obtained from the treatment of NK cells at a 6:1 ratio. (D and G) describe the 9:1 ratio activated aNK cell cytotoxic response.

Figure 5.

activated NK cells infiltrate the tumor spheroid at low e:t ratios. Time lapse of the central layers of CRC spheroids (green) obtained by confocal microscopy during the aNK (red) in vitro co-culture at 3:1 e:t ratio. After 24h in co-culture, aNK cells are able to infiltrate the tumor spheroid. This infiltration is observed till the 4^th day in which NK cells tend to migrate towards the periphery.