The combination of IFNγ and LPS is the strongest known stimulation for mouse iNOS expression and NO production in many cell types, but not in all. Moreover, the effects of this combination on EMMPRIN expression have not been explored. We used the macrophage-like cell line RAW 264.7 as a positive control (Figure 1) and compared it to the three mouse tumor cell lines, the renal (RENCA), colon (CT26), and prostate (TRAMP-C2) carcinoma cell lines. We show here that the TRAMP-C2 cells responded to the combined stimulation by increasing their iNOS mRNA and protein expression (22-folds, p < 0.01), as well as their NO production (17-folds, p < 0.001). By contrast, the CT26 colon tumor cells did not express the protein or produced nitrites (Figures 1A,C,D), similar to the RENCA cells (11), despite elevated iNOS mRNA levels (Figure 1E).

Since iNOS mRNA was increased in all three cell types, but protein expression was not, we reasoned that a post-transcriptional regulation of iNOS exists in CT26 and RENCA cells, but not in TRAMP-C2 cells. Indeed, the combined stimulation increased the expression of miR-146a-5p only in the RENCA and CT26 cells (by 34- and 7-folds, p < 0.01, Figure 1F). We also observed that the combined stimulation did not change the accumulation of EMMPRIN mRNA or protein in the three tumor cell lines (Figures 1B,G,H). Thus, the expression of iNOS is inversely correlated with miR-146a-5p expression in the three tumor cells, and EMMPRIN expression does not correlate to the stimulation or to miR-146a-5p expression, probably as it is already maximally expressed.

To demonstrate that miR-146a suppresses iNOS expression in CT26 tumor cells, we neutralized its activity by transfecting the cells with its antagomir, as we have done before in RENCA cells (11). We used the mirVana™ anti-miR-146a-5p inhibitor, a potent, chemically modified single-stranded RNA molecule with a sequence complementary to that of miR-146a-5p (anti-miR-146a-5p). The combined stimulation markedly elevated iNOS mRNA in both cell lines when transfected by either the antagomir or its negative control (p < 0.05, Figure 2B). However, the negative control did not induce iNOS protein expression or NO production in both cell lines, even in the presence of the combined stimulation, as evident by immunofluorescence (Figure 2C, red staining, and the relevant parts of Figure 2D) and nitrite accumulation (Figure 2A). Likewise, transfection with the antagomir in the absence of the combined stimulation did not induce iNOS expression (Figure 2A). Only transfection with the antagomir in the presence of the combined stimulation restored iNOS protein induction and NO production (threefolds and eightfolds for RENCA and CT26, respectively, p < 0.05, Figures 2A,C,D). Thus, iNOS expression and NO production in tumor cells require a strong pro-inflammatory stimulation, together with neutralization of miR-146a-5p activity, in both RENCA and CT26 cells.

To explore the effects of the combined stimulation and miR-146a-5p on EMMPRIN expression in the same transfected cells, we stained for EMMPRIN as well. EMMPRIN was constitutively expressed in both RENCA and CT26 cells, and no change was visible upon incubation with the combined stimulation. However, transfection of the antagomir resulted in a decrease in the intensity of EMMPRIN staining compared to the cells transfected with the negative control (Figure 2C, green staining, and the relevant parts of Figure 2D). This effect was also quantified by evaluating the amounts of the secreted protein (3-fold decrease for both cell lines, p < 0.05, Figure 3A), and by assessing the membranal expression of the protein by flow cytometry (1.5- to 2-fold decrease, p < 0.05, Figures 3B,C). However, EMMPRIN mRNA was unaffected by the combined stimulation or the transfection of the antagomir (Figure 3D). Thus, EMMPRIN expression is also post-transcriptionally regulated in both tumor cell lines.

Some tumor cells lose iNOS expression in order to escape immune-mediated death (10), and we have shown that despite the high levels of NO secreted by stimulated macrophages, they cannot kill RENCA cells that do not express iNOS, unless iNOS expression is restored by transfecting the cells with the miR-146a antagomir (11). When RENCA, CT26, or TRAMP-C2 cells were cocultured with the RAW 264.7 macrophages in the presence of the combined stimulation, only TRAMP-C2 cells exhibited increased death [48 ± 13% increase (Figure 4A), p < 0.001], despite the high NO levels accumulated in all cocultures (Figure 4B). Cell death was abrogated by the addition of the selective iNOS inhibitor 1,400W (p < 0.05), suggesting that it was NO dependent.

To show that the induction of death by pro-inflammatory macrophages depends on the activity of miR-146a in the tumor cells, we next cocultured RAW 264.7 macrophages with RENCA (Figure 4C) or CT26 (Figure 4E) cells transfected with anti-miR-146a, and assessed tumor cell death relative to cells transfected with the negative control. RENCA cell death was increased by 48 ± 6% (p < 0.001) and CT26 cell death was increased 25 ± 1.3% (p < 0.001), only when anti-miR-146a-5p was introduced and when the combined stimulation was present. Again, this was abolished by the (1400W) iNOS inhibitor, demonstrating an NO-dependent effect. Despite the difference in cell death, we did not detect a difference in nitrite accumulation between the anti-miR-146a-5p and anti-miR-NC transfected cells, suggesting that the macrophages contributed the bulk of nitrites (Figures 4D,F).

Changes in the angiogenic activity of EMMPRIN were detected by the concentrations of its induced pro-angiogenic factors VEGF and MMP-9 in the supernatants of transfected cells. In comparison to cells transfected with the negative control, marked reduction in MMP-9 levels (about 2-folds, p < 0.05, Figure 5A) and VEGF levels (about 34-folds, p < 0.05, Figure 5B) were observed after transfection of anti-miR-146a-5p, regardless of the presence of the combined stimulation. Likewise, relative to cells transfected with the negative control, the supernatants from cells transfected with the antagomir caused a 40% reduction (p < 0.05) in the number of closed lumens (Figure 6A), and a 20–30% inhibition (p < 0.05) in endothelial cell proliferation and migration in the wound assay (Figure 6B). In both assays, the combined stimulation had no additional effects.

To examine if the miR-146a-5p can be in vivo manipulated to reduce tumor size, we next subcutaneously implanted RENCA tumor cells in the syngeneic wild-type BALB/c mice. When tumors became palpable, we injected either the antagomir or its negative control to their circulation, with or without the simultaneous injection of RAW 264.7 cells that were previously in vitro stimulated with IFNγ and LPS for 24 h, to the rims of the tumors, where they would be least exposed to the hypoxic microenvironment. This was repeated three times every 7 days. Injection of the antagomir’s negative control (anti-miR-NC) with or without stimulated RAW264.7 cells did not affect tumor growth rate, and at the end of the experiment, the average tumor size was 1.54 ± 0.3 cm³ (Figure 7A). Injection of the antagomir alone resulted in a 1.4-folds reduction of tumor size (p < 0.05), whereas the combination of the antagomir and the stimulated macrophages resulted in a considerable slowing of the growth rate and about 6-folds reduction in tumor size (p < 0.001).

In mice injected with anti-miR-NC negative control, iNOS expression was not detected in the tumor cells, but macrophages that infiltrated the tumor after being injected to its rims expressed it in high levels, as evident by the intense staining (Figure 7D, low left panel). By contrast, iNOS expression was induced in RENCA tumor cells after anti-miR-146a-5p was injected i.v. (10-folds induction, p < 0.05, Figure 7D, right panels, Figure 7E).

EMMPRIN expression exhibited an inverse pattern to iNOS expression. Constitutive high expression levels of EMMPRIN were observed in the negative control group, and these were markedly reduced when anti-miR-146a-5p was injected, regardless of the injection of stimulated RAW 264.7 cells (about twofolds, p < 0.01, Figures 7B,C).

The effects of the treatment on angiogenesis were first estimated by the change in the mean vessel density (MVD) by staining for the endothelial marker CD31 (Figure 8A). Blood vessels in the negative control group injected with the anti-miR-NC were long, branched, and continuous (Figure 8A, top left panel), whereas in the group injected with both the anti-miR-146a-5p and stimulated macrophages, blood vessels were short, discontinuous, and with wider gaps between them (Figure 8A, bottom right panel). The vessel surface area, a measure of MVD, was gradually reduced (Figure 8B), culminating in a twofold decrease relative to the group receiving both anti-miR-146a-5p and stimulated macrophages (p < 0.001). A reduction in the levels of the pro-angiogenic factors VEGF (by 6.7-folds, p < 0.01, Figure 8C) and MMP-9 (by 5-folds, p < 0.05, Figure 8D) was observed in the tumor lysates between the groups receiving the anti-miR-NC and the group receiving anti-miR-146a-5p and stimulated macrophages.

The treatment with the antagomir and the stimulated macrophages reduced tumor cell proliferation (by twofolds, p < 0.05, Figures 9A,B) relative to the group receiving the anti-miR-NC alone, as assessed by the Ki-67 index. Complementarily, the rate of apoptosis was increased in this group, as evaluated by the TUNEL assay (2-folds, p < 0.01, Figures 9C,D) and the levels of activated caspase-3 (13-folds, p < 0.01, Figure 9E), relative to the negative control group.

Lastly, to detect immune-related changes in the TME, we measured the infiltration of CD8⁺ T cells. In mice receiving anti-miR-NC alone CD8⁺ T cells were few and mostly limited to the rims of the tumors (Figure 10A, top left panel). By contrast, in the group receiving both the antagomir and the stimulated macrophages many CD8⁺ T cells infiltrated the tumor tissue, resulting in an increase in the positively stained area (by fivefolds, p < 0.001, Figure 10A, bottom right panel, Figure 10B). Since we injected stimulated macrophages into the rims of the tumor, we saw no point in staining for their presence. However, nitrite concentrations, reflecting the macrophage mode of activation, were measured in the tumor lysates and showed an increase (3.7-folds, p < 0.05, Figure 10C) in the group receiving both the antagomir and stimulated macrophages, although the absolute levels were low. The same group also showed reduced levels of TGFβ, a dominant M2-related cytokine, relative to the other groups [by 5.7-folds (Figure 10D), p < 0.05]. Hence, we believe that these changes indicate immune modulation and the alleviation of immune suppression.

The tumorigenic mouse renal (RENCA, ATCC CRL-2947) and colon (CT26, ATCC CRL-2638) carcinoma cell lines were cultured in RPMI-1640 medium, 10% fetal calf serum (FCS), 1% l-Glutamine and antibiotics, with addition of 100 mM HEPES buffer (pH 7.4) for the RENCA cells, or 1% sodium pyruvate for the CT26 cells. The mouse TRAMP-C2 prostate cancer cell line (ATCC CRL-2731), the macrophage-like RAW 264.7 cell line (ATCC TIB-71), and the endothelial bEND3 cells (ATCC CRL-2299) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FCS, 1% l-glutamine and antibiotics, with addition of 5 µg/ml insulin and 10⁻⁸ mol/l methyltrienolone (R1881), the dihydrotestosteron analog (NLP005, Perkin-Elmer) for the TRAMP-C2 cells. All cell lines were used at passages 3–15 and regularly tested for morphological changes and presence of mycoplasma, RAW 264.7 cells were identified as macrophages by their ability to phagocytose zymosan particles, and tumor cells were tested as cells of epithelial origin by their expression of cytokeratin 18.

When indicated, cells were stimulated with IFNγ (100 U/ml, 485-MI-100, R&D systems, Minneapolis, MN, USA) and LPS (1 µg/ml, L-6529, Escherichia coli 055:B5, Sigma, St. Louis, MO, USA). To avoid immune stimulation or possible masking of signals by exogenous stimuli, cells were serum-starved before their exposure to the experimental conditions or their injection to mice.

Reverse transfection and inhibition of miR-146a-5p were performed exactly as before (11) for both RENCA and CT26 cells, only that the mirVana anti-miR-146a-5p inhibitor™ (4464084, Ambion, Austin, TX, USA) or its negative control (4464076, anti-miR-NC, Ambion), at 30 nmol/l each, were used instead of the first-generation inhibitors.

Quantitative real-time PCR analyses were performed as described before (11). Total RNA was extracted from 10⁶ RENCA or CT26 cells using the RNA extraction kit (17200, Norgen biotek, ON, Canada), and 500 ng of total RNA were transcribed to cDNA using the High Capacity cDNA Reverse Transcription kit (4368814, Applied Biosystems, Foster City, CA, USA). Expression of iNOS and EMMPRIN mRNAs and their reference gene PBGD, or miR-146a-5p and its reference gene U6 were determined by qPCR using TaqMan assay on demand kit with the StepOne system (Applied Biosystems) in triplicates according to the manufacturer’s instructions.

Determination of nitrites, western blots analyses, and cytotoxic assays were performed as before (11). The optical density of the bands in western blots was quantified using ImageJ. For the cytotoxic assays, the iNOS inhibitor 1,400 W (25 µM, W4262, Sigma) was used.

EMMPRIN expression was evaluated as before (17), using 1 µg of the FITC-conjugated anti-mouse CD147 or with its isotype control (123705, BioLegend, San Diego, CA, USA).

RENCA or CT26 cells (6 × 10⁴ cells) were transfected on cover slips with anti-miR-146a-5p as described above and fixed with cold methanol for 5 min at room temperature. Cells were permeabilized with 0.25% Triton-X 100 for 10 min, and incubated with blocking buffer (2% donkey normal serum, 0.1% Triton-X 100 in PBS) for 30 min at room temperature. Cells were stained with primary antibodies (rat anti-mouse EMMPRIN, MAB772, R&D systems, or rabbit anti-mouse iNOS, ab15323, Abcam, Cambridge, UK) diluted 1:250 in blocking buffer overnight at 4°C. Then secondary antibodies (Alexa 488-conjugated donkey anti-rat IgG, ab150153, Abcam, or Alexa 546-conjugated donkey anti-rabbit IgG, A10040, Thermo Fisher, Rockford, IL, USA) were diluted 1:500 in blocking buffer in the dark for 1 h at room temperature. Coverslips were mounted on a slide with fluoromount G. Three washes with PBS were applied after each step. Images were acquired by upright fluorescent trinocular microscope (Olympus BX-60, Tokyo, Japan) using the MS60 camera and the MShot Image Analysis System V1 (MSHOT, Guangzhou Micro-shot Technology Co., Guangzhou, China).

The mouse MMP-9, VEGF, TGFβ, and activated caspase-3 concentrations were determined as before (42). EMMPRIN concentrations were measured using with an ELISA kits (ab215405, Abcam) at a dilution of 1:200, according to the manufacturer’s instructions.

In vitro wound scratch assay was performed as described before (17), with the mouse bEND3 endothelial cell monolayers (10⁵ cells) seeded in 24-well dishes and incubated with experimental supernatants derived from RENCA or CT26 cells transfected with anti-miR-146a-5p or its negative control (diluted 1:2 with medium). Images of the field of injury were acquired at the beginning of the experiment and after 24 h. The average distances between the two sides of the wound were measured along the scratch (at least eight locations per field) in both time periods using the ImagePro plus 4.5 software (Media Cybernetics, Inc., Rockville, MD, USA), and the difference, which reflects the length to which the cells migrated, is presented.

Coultrex^® reduced growth factor basement membrane extract (40 μl/well, 3433, Trevigen, Gaithersburg, MD, USA) was used to coat 96-well plates at 4°C, and incubated at 37°C for 2 h to polymerize. bEND3 cells (8 × 10⁴) were seeded in triplicates in DMEM with 2% FCS and experimental supernatants diluted 1:2 with medium. After 6 h, the number of closed lumens per microscopic field, representing tube-like structures, was counted in two separate fields.

Experimental mouse model BALB/c mice (female, 8 weeks old, Envigo, Jerusalem, Israel) were kept with a 12 h light/dark cycle and access to food and water ad libitum. Tumors were generated as before (11), and when they became palpable at day 14, mice were randomly assigned to four groups that received the following treatments every 7 days: three i.v. injections of 0.025 mg/g body weight of (a) anti-miR-146a-5p or (b) anti-miR-negative control (anti-miR-NC) to the tail vein. Groups (c) and (d) were treated as groups (a) and (b), respectively, with the addition of 10⁶ RAW 264.7 cells stimulated with IFNγ (100 U/ml) and LPS (1 µg/ml) for 24 h, injected to the rims of the tumors. Tumors were measured every 3–4 days and their volume calculated (length × width × 0.5 cm³). At the end of the experiment, or when tumors were greater than 1.5 cm³, mice were euthanized and their tumor tissues were harvested. Part of the tumor was freshly frozen for evaluation of nitrite and cytokine concentrations in tumor lysates, while other parts were formalin-fixed and paraffin-embedded for later analysis by immunohistochemical staining.

Immunohistochemistry and assigning an immune reactive score was performed as described in Ref. (42). Antigen retrieval for iNOS was performed by microwave heating in citrate buffer pH 6.0, and the antibody used was rabbit anti-iNOS (Abcam). All sections were viewed under the bright field trinocular microscope (Olympus BX-60, Tokyo, Japan) and images were acquired with the MS60 camera and the MShot Image Analysis System V1 (MSHOT, Guangzhou Micro-shot Technology Co., Guangzhou, China). Vessel densities assessed by CD31 staining and by using a Weibel grid to calculate vessel surface area (48), and the fraction of Ki-67-positive tumor cells was calculated by the digital image analysis web application ImageJS (49). EMMPRIN and iNOS expression were assessed using the modified H-score, which assigns an immune reactive score on a continuous scale of 0–300, based on the percentage of positive cells expressing the protein at different intensities. Staining was divided into three categories: 1 for “light staining,” 2 for “intermediate staining,” and 3 for “strong staining.” The percentage of positive cells was determined according to the positive surface area of cells measured with ImagePro plus 4.5 software, and the score was calculated using the formula: 1 × (%1 positive cells) + 2 × (%2 positive cells) + 3 × (%3 positive cells).

All values are presented as means ± SE. Significance between two groups was determined using the two-tailed unpaired t-test. Differences between three or more experimental groups were analyzed using one-way analysis of variance (ANOVA) and the post hoc Bonferroni’s multiple comparison tests, and the two-way ANOVA following Bonferroni’s post-tests for comparing time and groups. P values exceeding 0.05 were not considered significant.