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

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.

Multiwell capillarity-based microfluidic device for the study of 3D tumour tissue-2D endothelium interactions and drug screening in co-culture models.

The tumour microenvironment is very complex, and essential in tumour development and drug resistance. The endothelium is critical in the tumour microenvironment: it provides nutrients and oxygen to the tumour and is essential for systemic drug delivery. Therefore, we report a simple, user-friendly microfluidic device for co-culture of a 3D breast tumour model and a 2D endothelium model for cross-talk and drug delivery studies. First, we demonstrated the endothelium was functional, whereas the tumour model exhibited in vivo features, e.g., oxygen gradients and preferential proliferation of cells with better access to nutrients and oxygen. Next, we observed the endothelium structure lost its integrity in the co-culture. Following this, we evaluated two drug formulations of TRAIL (TNF-related apoptosis inducing ligand): soluble and anchored to a LUV (large unilamellar vesicle). Both diffused through the endothelium, LUV-TRAIL being more efficient in killing tumour cells, showing no effect on the integrity of endothelium. Overall, we have developed a simple capillary force-based microfluidic device for 2D and 3D cell co-cultures. Our device allows high-throughput approaches, patterning different cell types and generating gradients without specialised equipment. We anticipate this microfluidic device will facilitate drug screening in a relevant microenvironment thanks to its simple, effective and user-friendly operation.

High-order TRAIL oligomer formation in TRAIL-coated lipid nanoparticles enhances DR5 cross-linking and increases antitumour effect against colon cancer.

During the last years, a great effort has been invested into developing new TRAIL formulations with increased bioactivity, trying to overcome the resistance to conventional soluble TRAIL (sTRAIL) exhibited by many primary tumours. In our group, we have generated artificial lipid nanoparticles decorated with sTRAIL (LUV-TRAIL), emulating the physiological TRAIL-containing exosomes by which T-cells release TRAIL upon activation. We already demonstrated that LUV-TRAIL has greater cytotoxicity against both chemoresistant haematologic tumour cells and epithelial carcinoma cells compared to a form of sTRAIL similar to that used in clinical trials. In this study we have tested LUV-TRAIL in several human colon cancer cell lines with different sensitivity to sTRAIL. LUV-TRAIL significantly improved sTRAIL cytotoxicity in all colon cancer cell lines tested. Trying to ascertain the molecular mechanism by which LUV-TRAIL exhibited improved cytotoxicity, we demonstrated that TRAIL-coated lipid nanoparticles were able to activate DR5 more efficiently than sTRAIL, and this relied on LUV-TRAIL ability to promote DR5 clustering on the cell surface. Moreover, we show that TRAIL molecules are arranged in higher order oligomers only in LUV-TRAIL, which may explain their enhanced DR5 clustering ability. Finally, LUV-TRAIL showed significantly better antitumour activity than sTRAIL in an in vivo model using HCT-116 xenograft tumours in nude mice, validating its potential clinical application.

TRAIL-coated lipid-nanoparticles overcome resistance to soluble recombinant TRAIL in non-small cell lung cancer cells.

PURPOSE: Non-small cell lung cancer (NSCLC) is one the types of cancer with higher prevalence and mortality. Apo2-Ligand/TRAIL is a TNF family member able to induce apoptosis in tumor cells but not in normal cells. It has been tested in clinical trials against different types of human cancer including NSCLC. However, results of clinical trials have shown a limited efficacy of TRAIL-based therapies. Recently we have demonstrated that artificial lipid nanoparticles coated with bioactive Apo2L/TRAIL (LUV-TRAIL) greatly improved TRAIL cytotoxic ability being capable of killing chemoresistant hematological cancer cells. In the present work we have extended the study to NSCLC. METHODS/PATIENTS: LUV-TRAIL-induced cytotoxicity was assessed on different NSCLC cell lines with different sensitivity to soluble TRAIL and on primary human tumor cells from three patients suffering from NSCLC cancer. We also tested LUV-TRAIL-cytotoxic ability in combination with several anti-tumor agents. RESULTS: LUV-TRAIL exhibited a greater cytotoxic effect compared to soluble TRAIL both in A549 cells and primary human NSCLC cells. LUV-TRAIL-induced cell death was dependent on caspase-8 and caspase-3 activation. Moreover, combination of LUV-TRAIL with other anti-tumor agents such as flavopiridol, and SNS-032 clearly enhanced LUV-TRAIL-induced cytotoxicity against NSCLC cancer cells. CONCLUSION: The novel formulation of TRAIL based on displaying it on the surface of lipid nanoparticles greatly increases its anti-tumor activity and has clinical potential in cancer treatment.