Despite NK cells being originally identified because of their ability to kill tumor cells in vitro, only limited information is available on NK cells infiltration of malignant tumors, especially in humans. NK cells infiltrating human colorectal carcinomas (CRCs) were analyzed to identify their potential protective role in an antitumor immune response. The expression and function of relevant molecules were analyzed from different sources, comparing tumor-associated NK cells (TANKs) with autologous peripheral blood NK cells (PB-NKs) from CRC patients-the latter in comparison with PB-NKs from normal donors. TANKs displayed a profound alteration of their phenotype with a drastic reduction of NK cell receptor expression. Co-culture experiments showed that CRC cells produce modulation in NK phenotype and functionality. Moreover, PB-NKs from CRC patients also exhibited an altered phenotype and profound defects in the ability to activate degranulation and IFN-γ production. For the first time, TANK and PB-NK cells from CRC patients have been characterized. It is shown that they are not capable of producing relevant cytokines and degranulate. Taken together, our results suggest that NK cells from CRC patients present alterations of phenotype and function therefore supporting the progression of cancer.

The clinical outcome of colorectal cancer (CRC) is associated with the immune response; thus, these tumors could be responsive to different immune therapy approaches. Natural killer (NK) cells are key antitumor primary effectors that can eliminate CRC cells without prior immunization. We previously determined that NK cells from the local tumor environment of CRC tumors display a profoundly altered phenotype compared with circulating NK cells from healthy donors (HD). In this study, we evaluated peripheral blood NK cells from untreated patients and their possible role in metastasis progression. We observed profound deregulation in receptor expression even in early stages of disease compared with HD. CRC-NK cells displayed underexpression of CD16, NKG2D, DNAM-1, CD161, NKp46, and NKp30 activating receptors, while inhibitory receptors CD85j and NKG2A were overexpressed. This inhibited phenotype affected cytotoxic functionality against CRC cells and interferon-γ production. We also determined that NKp30 and NKp46 are the key receptors involved in detriment of CRC-NK cells’ antitumor activity. Moreover, NKp46 expression correlated with relapse-free survival of CRC patients with a maximum follow-up of 71 months. CRC-NK cells also exhibited altered antibody-dependent cellular cytotoxicity function responding poorly to cetuximab. IL-2 and IL-15 in combination with cetuximab stimulated NK cell, improving cytotoxicity. These results show potential strategies to enhance CRC-NK cell activity.

Triple negative breast cancer (TNBC) patients are not likely to benefit from anti-estrogen or anti-HER2 therapy and this phenotype is associated with a more aggressive clinical course and worse clinical outcome. Taking into account the limited treatment possibilities in TNBC, the aim of the present work was to study a potential therapy based on Cetuximab-mediated immune activity by natural killer (NK) cells. We performed in vitro studies on human breast cancer (BC) cell lines, IIB-BR-G, and the in vivo metastatic variant IIB-BR-G MT. The immunohistochemical analysis showed a TNBC phenotype with high but different levels of EGFR expression on each cell line, measured by flow cytometry. DNA sequencing showed that both cell lines have a mutated K-RAS status, 38 G > A at codon 13. Consequently, Cetuximab did not inhibit cellular proliferation or induce apoptosis. We investigated if Cetuximab could trigger immune mechanisms, and we determined that both cell lines treated with 1 μg/ml Cetuximab were susceptible to antibody dependent cellular cytotoxicity (ADCC), mediated by peripheral blood mononuclear cells (PBMC). At 50:1 effector:target ratio, lytic activity was 34 ± 2% against IIB-BR-G and 27 ± 6% against IIB-BR-G MT cells. PBMC pretreatment with IL-2 allowed reaching 65 ± 3% of Cetuximab-mediated ADCC against IIB-BR-G and 63 ± 6.5% against IIB-BR-G MT. Furthermore, IL-15 pretreatment increased the ADCC up to 71 ± 3% in IIB-BR-G and 79 ± 3.5% in IIB-BR-G MT. We suggest that NK cells are the effectors present in PBMC since they were able to induce ADCC at lower effector:target ratios. Besides, IL-2- and mainly IL-15-induced upregulation of NK activating receptors CD16 and NKG2D and enhanced IFN-γ production. EGFR-expressing TNBC could be killed by Cetuximab-mediated ADCC at clinically achievable concentrations. IL-15 could advantageously replace IL-2 in most of its immunologic activities, stimulating the ability to produce IFN-γ, and paralleling the up-regulation of activating receptors.