Inhibition of TGF-β signaling increases intra-tumoral CD11b+ cells that express neutrophil (Ly6G+) rather than macrophage (Ly6G−) markers
To evaluate the role of myeloid (CD11b+) cells, mice bearing established flank tumors from three syngeneic models were fed with chow containing SM16 or control chow. Tumors were harvested and subjected to FACS to detect CD11b+ cells and different myeloid cell markers.
As shown in , administration of SM16 increased the percentage of CD11b+
cells in the tumors by 30−45% (p <0.02). To differentiate macrophages from neutrophils, we used the 1A8 anti-Ly6G antibody which is found only on neutrophils (Daley et al., 2008
). SM16 treatment led to significant increases in the percentage of intra-tumoral Ly6G+
cells and only minor changes in the Ly6G−
cells (mostly macrophages). As seen in , virtually all the Ly6G+
cells were also CD11b+
SM16 causes an influx of CD11b+ Ly6G+ granulocytic cells into tumors
To ask if neutrophils travel to areas of tumor necrosis, we performed immunohistochemistry of tumors using the Ly6G antibody. We found an increased number of Ly6G+
cells in tumors from SM16-treated mice and that the cells were primarily in the non-necrotic areas of the tumors (Supplemental Fig. 1
). We also blocked TGF-β activity using a neutralizing anti-TGF-β monoclonal antibody (1D11) in the AB12 cell line and confirmed significantly increased levels of intra-tumoral neutrophils (CD11b+
) (data not shown).
Evaluation of myeloid cell populations in the spleens of mice treated with SM16 versus control showed no significant changes in the percentage of CD11b+ cells (12.1 ± 4.7 in control-treated vs. 13 ± 0.7 in SM16-treated mice), CD11b+/GR1+ myeloid derived suppressor cells (10.7 ± 4.3 vs. 11.7 ± 0.7) or CD11b+/Ly6G+ cells (9.2 ± 3.8 vs. 9.6 ± 0.6). There was no change in the percentage of CD11b+/Ly6G+ neutrophils in the blood in control tumor-bearing mice (41.3% of leukocytes) versus SM16 treated mice (38.3% of leukocytes). The percentage of CD11b+/Ly6G− in the blood was negligible in both groups of mice. These data suggest that the changes in TAN were not systemic, but rather due to a change in recruitment and/or persistence within the tumors.
To evaluate the morphology of the TAN, intra-tumoral CD11b+/Ly6G+ cells were isolated. As seen in , the Ly6G+ cells isolated from flank tumors from both control untreated mice and SM16-treated mice had a clear neutrophil-like morphology. Interestingly however, most of the neutrophils in the SM16-treated tumors were more lobulated and hyper-segmented (bottom panel), losing some of the characteristic circular nuclei appearance typical of blood or bone marrow murine neutrophils (top panel), and relatively maintained in control TAN (middle panel),.
The morphology of TAN in control and SM16-treated mice compared to bone-marrow neutrophils
We further evaluated the pulmonary influx of CD11b+ cells in the orthotopic transgenic activated K-ras model of bronchogenic adenocarcinoma of the lung. Eight to nine weeks after activation of the K-ras mutation, we treated the mice with SM16 or control chow followed by flow cytometry of the whole lung. As seen in , we found a 43% increase in the percentage of neutrophils in the lungs of the SM16 mice (8 ± 0.5) compared to the control mice (5.6 ± 0.9) (p=0.03). Similar to the results in the flank models, the percentage of CD11b+/Ly6G− cells in control (2.8 ± 0.7) vs. SM16-treated (2.1 ± 0.5) mice did not increase.
TGF-β-blockade increases the mRNA for neutrophil chemoattractants
We next used real-time RT-PCR to measure the level of cytokines, chemokines, and cell adhesion molecules in flank tumors derived from AB12, LKR and TC1 cells. As we have shown previously (Kim et al., 2008
; Suzuki et al., 2007
), SM16 treatment resulted in changes in the tumor microenvironment manifested by increased levels of iNOS, cytokines and cell adhesion molecules, along with decreased levels of arginase ().
Real-Time RT-PCR analysis of whole tumors and neutrophil subsets with and without treatment with SM16
We also assessed the message levels of selected chemokines that have an established role in the recruitment and chemoattraction of neutrophils (Kobayashi, 2008
). As shown in , we found a significant increase in the mRNA levels of 3 potent neutrophil chemoattractants: MIP-2α/CXCL2, LIX/CXCL5 and MIP1α/CCL3. We also found a 2 to 3-fold increase in two other neutrophil chemoattractants in 2 of the lines – Rantes/CCL5 (in AB12 and LKR) and KC/CXCL1 (in AB12 and TC1), as well as a 2-fold increase in the CXCL1 protein in AB12 tumors (data not shown). In the TC1 tumor, but not in the AB12 tumor, we found a significant 7-fold increase in GM-CSF – another known neutrophil chemoattractant (Wang et al., 1988
). To evaluate a potential source of these chemokines, we isolated mRNA from TAMs of AB12 tumors. We found a 2.5 to 3-fold increase in the mRNA levels of CXCL2 and CXCL5 in isolated macrophages (CD11b+
) of tumors from SM16-treated mice, with no change in CXCL1 levels. These findings suggest that the blockade of TGF-β by SM16 induces secretion of neutrophil chemoattractants, at least partially from tumor macrophages, and expression of adhesion molecules, both of which could augment the recruitment of neutrophils into the tumor.
Intra-tumoral neutrophils are important effector cells in the anti-tumor effect of anti-TGF-β receptor treatment
We next studied the functional significance of TAN in the AB12 model by depleting the Ly6G+ cells in untreated and SM16-treated tumor-bearing animals.
We first injected the 1A8 antibody or isotype-matched control IgG intraperitoneally. A significant reduction of 80−90% in systemic and intra-tumoral neutrophils was noted by FACS in these experiments (data not shown). Depletion of neutrophils in non-SM16 treated mice resulted in a small reduction in tumor growth (). In contrast, depletion of neutrophils in SM16-treated mice resulted in a significantly reduced effect of SM16 (); that is, the tumors grew more rapidly when neutrophils were depleted in the SM16-treated mice. Interestingly, when the systemic neutrophils returned in 3−4 days after the last injection, the growth inhibition with SM16 treatment appeared to also return (, last time point).
Effect of neutrophil depletion on tumor growth and on tumor response to SM16
To determine if local depletion of neutrophils had a similar effect, we injected the antibodies at lower dose, directly into the tumors using a previously described approach (Chen et al., 2007
; Yu et al., 2005
) (). Using flow cytometry, we confirmed a 70−80% reduction in the percentage of intra-tumoral Ly6G+
cells. Local depletion of neutrophils in non-SM16-treated animals resulted in a small but significant slowing of tumor growth (p < 0.05) (). Again, in contrast, when animals were treated with SM16 and also had neutrophils depleted, the SM16-induced treatment effect was significantly blunted. Treatment with SM16 reduced tumor burden by 80−90% compared to controls in the non-depleted mice, but tumor size was reduced by only 40% compared to controls in the mice that received SM16 plus repeated intra-tumoral depletion of Ly6G+
cells (). It should be noted that after intra-tumoral injection, we also found a 30−50% reduction in blood neutrophils (data not shown), suggesting that the antibody may also have a systemic effect. Together, these data indicate that depletion of neutrophils causes a significant reduction in the anti-tumor effect of TGF-β blockade, suggesting that neutrophils contribute to the antitumor activity of TGFβ blockade.
Inhibition of TGF-ß signaling in vivo increases CD11b+ cell cytotoxicity via an oxygen radical-dependent mechanism
To evaluate the cytotoxic potential of the intra-tumoral myeloid cells, microbeads were used to purify CD11b+ cells from AB12 tumors resulting in more than 90% purity (by FACS) in all experiments (data not shown). The isolated CD11b+ cells were co-cultured at varying ratios with luciferase-labeled AB12 tumor cells and the number of viable tumor cells was determined after 24 hours. CD11b+ cells from untreated control mice were found to be non-cytotoxic up to a ratio of 20:1 (macrophage to tumor cell), while CD11b+ cells isolated from the tumors of SM16-treated mice showed dose-dependent cytotoxicity, with significantly more killing than control myeloid cells at ratios of 10:1 and 20:1 (). shows data from six independent experiments with a CD11b+/tumor ratio of 20:1. The average killing induced by co-culture of CD11b+ cells from SM16-treated mice with AB12 cells was 41.6% vs. 16.3% in co-culture with CD11b+ cells from control mice (p=0.00013). Similar results were found with evaluation of LKR tumor-cells cytotoxicity, assessed by LDH levels (data not shown).
SM16 CD11b+ cell cytotoxicity is primarily due to Activated Neutrophils, via an oxygen radical-dependent mechanism
To evaluate possible mechanisms of killing by the intra-tumoral CD11b+ cells, tumor explants were isolated and put in medium for 24 hours. Tumors from SM16-treated mice secreted 40% higher levels of NO (p=0.08, ). In contrast, we found no change in the secretion of TNF-α (). Furthermore, the level of hydrogen peroxide (H2O2) secretion in PMA-activated CD11b+ cells from SM16 treated-tumors was 45% higher than in CD11b+ cells isolated from control mice (p<0.05) (). Moreover, there was no reduction in tumor cell killing in the presence of either anti-mouse TNFα antibodies or N-methyl-arginine (NMA), an inhibitor of NO synthase (iNOS). In contrast, the blockade of superoxide and H2O2 by the addition of superoxide dismutase (SOD) and catalase (Cat) respectively, significantly reduced the cytotoxicity of the CD11b+ cells to tumor cells, reducing the killing to 20.8% (p=0.0039), a level lower than the killing induced by control CD11b+ cells. These data show an important role for oxygen radicals in this in vitro assay of myeloid cell-induced tumor cytotoxicity.
CD11b+ cell cytotoxicity is primarily due to activated neutrophils
CD11b+ cells were next sorted using flow cytometry to separate the tumor-associated macrophages and tumor-associated neutrophils. We confirmed that the percentage of Ly6G+ cells (by flow cytometry) and neutrophilic-like cells (by morphologic appearance) in the isolated neutrophils was >95% following isolation. Control TAM, Control TAN, SM16 TAM, and SM16 TAN were co-cultured for 24 hrs with AB12 cells and cytotoxicity to tumor cells assessed. At ratios equal or lower than 10 viable neutrophils/macrophages to each tumor cell, there was no significant killing by either TAM or TAN (data not shown). Even at a ratio of 20 macrophages per tumor cell, TAM from control mice were mildly supportive to tumor growth. TAM from SM16-treated mice, and TAN from control mice had small amounts of killing capacities (up to 10%). The only cells able to induce significant tumor cytotoxicity were the TAN from the SM16-treated mice, which at a ratio of 20 to 1, showed a killing rate similar to whole CD11b+ cells (about 40% killing) (). These data show that the tumor-associated neutrophils make a significant contribution to the direct tumor cytotoxicity of myeloid cells following TGF-β blockade.
We further evaluated the expression of surface expressed “killing” molecules on TAN and found low expression of FAS-ligand and TRAIL by flow cytometry on TAN from both the control and SM16-treated mice. However, the percentage of FAS+ neutrophils increased in the tumors from SM16-treated mice by 25 % (14.3% ± 1.3 of neutrophils in control mice vs. 18.3% ± 1.4 in SM16-treated mice, p<0.05), with a similar increase in the mean fluorescence intensity (MFI) of FAS in the neutrophils (31.4 ± 2.5 vs.37.2 ± 1.4, p=0.05).
The TAN present after TGF-β-blockade have a more immunostimulatory mRNA profile than TAN from untreated mice
To study other phenotypic change in the neutrophils following TGF-β blockade, we performed real time RT-PCR of selected receptors, chemokines and cytokines in TAN. For comparison, we also examined mRNA levels in neutrophils isolated from the bone marrow of naïve mice and tumor-associated macrophages (CD11b+/Ly6G− cells) from control, tumor-bearing mice. We normalized all values to the levels found in TAN from control animals ().
Bone marrow neutrophils had undetectable levels of arginase, iNOS, CCL3, CCL5, and CCL2, whereas (with the exception of iNOS) these genes were highly expressed in control TAN from AB12 tumors (left panel, ). Unlike many of the other cytokines examined, however, TNFα and VEGF message levels were detected in bone marrow neutrophils from naïve mice at similar levels as in control TAN. A similar pattern was seen in LKR tumors (right panel, ), except that basal VEGF levels in bone marrow neutrophils were only 10% of those found in TAN.
Of even more interest was the effect of TGF-β blockade on TAN gene expression. In both types of tumors, control TAN were found to have high levels of arginase, an important immunosuppressor of the adaptive immune system (Rodriguez and Ochoa, 2008
). In fact, the levels of arginase message in control TAN were equal to or even higher than in control tumor associated macrophages. However, arginase levels were 2 to 5-fold lower in TAN from mice treated with SM16. In contrast, the level of TNF-α mRNA, an important immunostimulator, was significantly increased with TGF-β blockade by more than 7-fold in AB12 TAN and 2.4-fold in TAN from LKR tumors. The levels of CCL2 and CCL5 were significantly lower in SM16-treated TAN (AB12 tumors), whereas CCL3 was 2 to 2.7-fold higher in AB12 or LKR-bearing SM16-treated mice. ICAM-1 mRNA levels were markedly increased in SM16-treated neutrophils (6.7 to 36.4-fold). The level of ICAM-1 surface expression was increased by about 2-fold in the neutrophils of SM16-treated mice, as shown by flow cytometry (7.4% ± 0.1 of neutrophils in control mice vs. 12.6% ± 0.8 in SM16-treated mice, p<0.01). mRNA for VEGF, was reduced by 2-fold in the LKR SM16-treated tumors, but had only a non-significant trend to reduction in the AB12 tumor.
We also analyzed neutrophils isolated from lungs of tumor-bearing mice with advanced tumors from the orthotopic K-ras model and found very similar differences between the neutrophils in the SM16-treated mice and those from the control, untreated mice ().
Anti-tumor activity, as well as neutrophil recruitment to the tumors of SM16-treated mice, is dependent on the presence of CD8+ T cells
Given that systemic CD8+
T cell depletion essentially eliminates the effect of TGF-β blockade on tumor growth (Suzuki et al., 2007
) and our data showing that the anti-tumor effect was also partially neutrophil dependent (), we wanted to explore the intra-tumoral connections between TAN and CD8+
We first evaluated the effect of CD8+ T cell depletion on the phenotype of the tumor-associated myeloid cells. Animals bearing AB12 tumors were treated with SM16 with or without CD8+ T cell depletion. Systemic administration of anti-CD8 antibody resulted in depletion of more than 90% of CD8+ cells from the spleen (data not shown). As shown in (and similar to our previous data), all of the anti-tumor effect of SM16 was lost with CD8+ T cell depletion. Given complete loss of efficacy, we did not try to combine depletion of CD8 and depletion of neutrophils in the SM16 treated animals. However, we did study combined depletion of CD8 cells and neutrophils in control, untreated mice. As shown in (left columns), and similar to our findings in , intra-tumoral depletion of neutrophils led to a slowing of tumor growth. However, even though the tumors grew faster (as expected after CD8 depletion), we noted that the mild, but significant, anti-tumor effect of neutrophil depletion was maintained (, right columns). These data suggest that the neutrophils do have some pro-tumor effect that is independent of the presence of CD8 CTLs.
CD8+ cell depletion blocks all of the SM16 clinical effect and reduces influx of neutrophils to the tumors
Consistent with the data shown in , the percentage of CD11b+/Ly6G+ cells in the non-CD8 depleted tumors increased with SM16 treatment from 3.6% to 6.4% (p<0.05) (). CD8 depletion, by itself, reduced the percentage of neutrophils in the tumor to 1.7% of the cells (p<0.05). Recruitment of neutrophils after treatment with SM16 in the CD8+ T cell-depleted mice was somewhat blunted, increasing to 2.4% of the cells, however, this was less than control untreated, non-depleted tumors ( and individual panels in ). This small increase in the percentage of neutrophils was not sufficient to induce an anti-tumor growth effect, as noted in .
Depletion of neutrophils affects the activation of CD8+ CTLs
To understand the effect of TAN on CD8+
T cell activation, we evaluated the expression of two established surface activation markers, 4−1BB (CD137) and CD25. Tumor-bearing animals were left untreated or given SM16 chow with or without neutrophil depletion. Tumors were harvested one week after SM16 treatment and subjected to FACS. As we have previously shown (Kim et al., 2008
; Suzuki et al., 2007
; Wallace et al., 2008
), SM16 treatment led to activation of CD8+
T cells compared to controls, with the percentage of cells positive for 4−1BB rising from 35.1% to 65.8% of the CD8+
T cells (p<0.05) and the MFI of 4−1BB increasing from 18.1 to 42.6 (p<0.01) ().
Neutrophil depletion increases CD8+ T-cell activity in untreated mice, but reduces CD8+ T-cell activity in SM16-treated mice
Ly6G+ depletion slightly increased the percentage of intra-tumoral CD8+ cells in control mice (NS), and did not change this percentage in SM16-treated mice (data not shown). However, depletion of neutrophils in control mice (having a “pro-tumor” phenotype) led to significantly increased CD8+ T cell activation: 72.6% of the cells were found to be 4−1BB+ in tumors from neutrophil-depleted, untreated mice vs 35.1% in control mice (p<0.01, ). The MFI of 4−1BB in these CD8+ T cells increased from 18.1 to 36.9 in control versus depleted mice (p<0.01, ). This increased activation of CD8+ cells in control tumor-bearing mice following neutrophil depletion is consistent with the decreased tumor growth in neutrophil-depleted mice ().
We then studied the effect of neutrophil depletion on CD8+ T cell activation in SM16-treated animals. Depletion of these neutrophils (having an “anti-tumor” phenotype) led to significant decreases in CD8+ T cell activation. The percentage of 4−1BB+ activated cells was reduced from 65.8% of the CD8+ T-cells to only 49.7% (p<0.01, ), and the MFI was reduced from 42.6 to 18.7 (p<0.01, ). These results suggest that neutrophil depletion impairs both SM16-induced activation of CD8+ T cells and SM16-mediated tumor growth inhibition (see ). Similar results of CD8 activity were found when the percentage of CD8+/CD25+ was evaluated (data not shown).