NK cells in the tumor microenvironment

Abstract

The presence of natural killer (NK) cells in the tumor microenvironment correlates with outcome in a variety of cancers. However, the role of intratumoral NK cells is unclear. Preclinical studies have shown that, while NK cells efficiently kill circulating tumor cells of almost any origin, they seem to have very little effect against the same type of tumor cells when these have extravasated. The ability to kill extravasated tumor cells is, however, is dependent of the level of activation of the NK cells, as more recent published and unpublished studies, discussed below, have demonstrated that interleukin-2-activated NK cells are able to attack well-established solid tumors.

FIG. 1. Accumulation of IL-2–activated NK (A-NK) cells selectively at tumor sites

Flow-sorted NKp46+ splenocytes from congenic Thy1.1+ C57BL/6 mice were cultured with IL-2 for 5 days and injected i.v. into C57BL/6 mice (Thy1.2+) with 9-day-old B16 tumors. Each mouse received 5 million A-NK cells. 30,000 IU Peg–IL-2 was injected i.p. every 12 h (max. six injections). Organs were removed at 72 h after injection of the A-NK cells and fresh frozen. Eight micron cryosections were all stained with PE-conjugated anti-Thy1.1 antibodies (NK cells begin to express Thy1 within 24 h of IL-2 activation). Some sections were also stained with FITC-conjugated anti-laminin antibodies. (A) DIC picture of lung tissue with multiple black-pigmented B16 melanoma metastases. (B) Fluorescent photomicrograph of the same sections as in (A), showing a dense accumulation of PE-Thy1.1+ A-NK cells (red dots) selectively in the black-pigmented metastases. White arrow points to a single PE-Thy1.1+ A-NK cell. (C) and (D) same as (A) and (B), respectively, but at higher magnification and with staining of laminin (green fluorescence in (D)). Note the strong preference of the A-NK cells for the tumor tissue. (E) and (F) show a DIC and a fluorescent picture, respectively, of laminin-stained ovarian tissue (green in (F)) with a black-pigmented B16 metastasis infiltrated by PE-Thy1.1+ A-NK cells. Bars in A–B = 200 μm, Bars in C–F = 100 μm.

FIG. 2. In vivo proliferation increases the number of transferred A-NK cells found at tumor sites

Flow-sorted NKp46+ splenocytes from congenic Thy1.1+, CD45.2+ and congenic Thy1.2+, CD45.1+ C57BL/6 mice were cultured with IL-2 for 5 days and injected i.v. into C57BL/6 mice (Thy1.2+,CD45.2) with 9-day-old B16 tumors. Before injection either the Thy1.1+ or the CD45.1+ A-NK cells were irradiated (450 rad). Each mouse received a mixture of 2.5 million Thy1.1+ and 2.5 million CD45.1+ A-NK cells. 30,000 IU Peg–IL-2 was injected i.p. every 12 h (max six injections). Organs were removed at 72 h after injection of the A-NK cells and were fresh frozen. (A) B16 lung metastasis from an animal receiving non-treated Thy1.1+ and irradiated CD45.1+ A-NK cells. While many Thy1.1+ A-NK cells (stained with FITC-anti-Thy1.1 antibodies) are infiltrating the tumor, only few of the irradiated CD45.1+ A-NK cells (stained with PE-anti-CD45.1 antibodies) can be observed. (B) Close-up of a B16 lung metastasis from an animal injected with irradiated Thy1.1+ and non-treated CD45.1+ A-NK cells. While few of the irradiated Thy1.1+ A-NK cells (stained with FITC-anti-Thy1.1 antibodies) are infiltrating the tumor, many non-irradiated CD45.1+ A-NK cells (stained with PE-anti-CD45.1 antibodies) can be observed throughout the tumor nodule. Bars = 50 μm.

FIG. 3. Tumor-infiltrating A-NK cells eliminate tumor cells

Five million Thy1.1+ A-NK cells were injected into Thy1.2+ C57BL/6 mice bearing 9-day-old B16 melanoma lung metastases. 30,000 IU Peg–IL-2 was injected i.p. every 12 h (max. six injections). Organs were removed at 16 and 120 h after injection of the A-NK cells and were fresh frozen. (A) DIC picture of a B16 lung tumor from an animal injected with A-NK cells 16 h earlier. (B) Fluorescence picture of the tumor shown in (A) after staining with a 1:1 mixture of polyclonal rabbit anti-Tyrp114,15 and Pmel1716,17 antibodies (kindly provided by Dr. V. Hearing, NIH) and subsequently with FITC-conjugated anti-rabbit antibody. To reveal A-NK cells, the section was also stained with PE–anti-Thy1.1 antibodies. At this point in time, relatively few A-NK cells are found in the tumor, which is composed of B16 tumor cells (shoulder-to-shoulder). (C) DIC picture of B16 lung tumors from an animal injected with A-NK cells 120 h earlier. (D) Same area as in (C). Note that, while the tumor on the left is heavily infiltrated by A-NK cells (PE-Thy1.1+), which have almost replaced the tumor cells (FITCE+), the tumor on the right contains only few infiltrating A-NK cells but many tumor cells. Bars = 100 μm.

FIG. 4. Tumor-infiltrating A-NK cells depend on IL-2 for survival

GFP+ and GFP+,IL-2+ A-NK cells were produced by stable transduction of Mau-1 cells (a long-term A-NK-cell line (C57BL/6) developed in our lab) with adeno-associated virus containing the gene for GFP or the genes for both GFP and IL-2. (A) DIC picture of a B16 lung tumor from an animal injected i.v. with 5 million IL-2- and GFP-producing Mau-1 cells 72 h earlier. (B) Fluorescence picture of the tumor shown in (A) showing numerous GFP+ cells infiltrating the tumor. (C) DIC picture of a B16 lung tumor from an animal injected i.v. with 5 million GFP-producing Mau-1 cells 72 h earlier. (D) Fluorescence picture of the tumor shown in (A) showing very few GFP+ cells infiltrating the tumor. No exogenous IL-2 was given to support the injected Mau-1 A-NK cells. Bars = 100 μm.

FIG. 5. Viral infection induces a vigorous tumor infiltration by host NK cells

Mammary brain and leptomeningeal metastases were induced by injection of Her2neu expressing D2F2/E2 cells into the cisterna magna (CM) of Balb/c mice. Nine days later, Her2-targeting VSV were injected into the CM. Three days later, brains were removed and sections of brain were stained with NKp46 antibody to reveal host NK cells. (A) Fluorescence picture of a Hoechst 33342-stained brain section with a large D2F2/E2 tumor (the nuclei-dense lower half of the picture) from an animal receiving Her2-targeting VSV 3 days earlier. (B) Same area as (A), showing a high number of PE-NKp46+ host NK cells infiltrating the D2F2/E2 tumor (below the white line). Note the low density of host NK cells in the normal brain tissue (above the white line). (C) and (D) show an D2F2/E2 tumor from another animal receiving Her2 targeted VSV 3 days earlier (to the right of the white line). Note the dense cluster of host NK cells to the right and the absence of host NK cells from the adjacent normal brain tissue (to the left of the white out-line). (E) and (F) show an D2F2/E2 tumor (below white out-line) from a control animal, which did not receive the Her2-targeted virus. Note the very low number of host NK cells in the tumor tissue. Bars = 200 μm.