Results

Regressing skin tumours undergo progression when transfected with the gene for TGF

The PGW skin tumour is a clone of the polyclonal UV13.1 cell line that has been transfected with an empty gene vector. This cell line produces very little TGF (Figure 1a) and undergoes regression when injected subcutaneously into the backs of syngeneic C3H/HeN mice (Figures 1b and c). Transfection of this cell line with the gene for TGF (PGWTGF) results in the production of significantly increased amounts of TGF (Figure 1a), and the ability to evade immunological destruction and grow progressively (Figures 1b and c), as shown by the significantly increased tumour diameter and incidence.

Figure 1.

TGF-transfected tumour cells (PGWTGF) secrete elevated levels of TGF and undergo progressive tumour growth in vivo. (a) TGF production was assessed in supernatant collected from in vitro-cultured tumour cells using an enzyme-linked immunosorbent assay kit, mean+s.e.m. from four replicates shown. (b) Pooled tumour growth (mean diameters.e.m.) and (c) tumour incidence measurements from five independent experiments, PGW; n=16 mice and PGWTGF; n=48 mice (where s.e.m. is not obvious it is too small to be observed in this scale). Fully regressed tumours were included as zero for the calculation of mean diameter. ^*P<0.05. TGF, transforming growth factor-₁.

Full figure and legend (25K)

TGF secretion decreases dendritic cells and increases macrophage infiltration into the tumour

Four-colour flow cytometry enabled the identification of tumour-infiltrating CD45⁺CD11c⁺CD11b⁺MHC II^high DC and CD45⁺CD11c^-CD11b⁺MHC II⁺ TAM (Figure 2a). CD11c⁺ DC from both tumour groups expressed significantly higher levels of MHC II (over twofold higher mean fluorescence intensity) than CD11c^-CD11b⁺ macrophages (Table 1). A detailed flow cytometric phenotypic analysis of these APC subsets showed no consistent difference in the expression of the chemokine receptors CCR6 or CCR7 nor in the expression of the costimulatory molecule CD80 (Table 1). CD86 analysis revealed that TAM infiltrating PGWTGF tumours expressed significantly lower levels of CD86 compared to DC infiltrating PGW tumours (Table 1; P<0.05). Analysis of the ratio of DC to TAM demonstrated that transfection with TGF not only decreased the percentage of tumour-infiltrating DC but increased the percentage of TAM (Figure 2b), however there was about fivefold greater proportion of DC than macrophages.

Figure 2.

Progressor skin tumours are infiltrated by a significantly greater percentage of macrophages while regressor tumours are populated by dendritic cells. (a) The first column of dot plots show the level of isotype staining on whole tumour cell preparations compared to the level of CD45 and CD11c staining (second column). Four-colour flow cytometry identified CD11c⁺CD45⁺ DC (R1) and CD11c^-CD45⁺ non-DC inflammatory cell infiltrates (R2). The third column of dot plots show the level of isotype staining for CD11b and MHC II on CD45⁺ cells compared to the expression of CD11b and MHC II on DC (R1; fourth column) and non-DC inflammatory cells (R2; last column of dot plots). TAM were identified as CD11c^-CD45⁺CD11b⁺ (last column in (a)). Dot plots are representative of four separate experiments. (b) The percentage of CD11c⁺CD45⁺ CD11b⁺ DC or CD11c^-CD45⁺CD11b⁺ TAM from four separate experiments were pooled. Mean+s.e.m. shown. ^*P<0.05. DC, dendritic cells; TAM, tumour-associated macrophages.

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Table 1 - Phenotype of tumour-infiltrating antigen-presenting cells.

Full table

TGF alters the APC ratio and enhances macrophage phagocytosis in vitro

To investigate further the effect of TGF on APC number and function, we isolated and incubated whole spleen cells with various concentrations of recombinant TGF. Adding TGF to the culture media resulted in a greater than twofold reduction in the percentage of CD11c⁺CD11b⁺ DC and a concordant increase in the percentage of CD11^-CD11b⁺ macrophages (Figure 3a). The TGF doses used in these experiments (0.2–1.0 ng ml^-1) approximate the levels of TGF secretion by progressor PGWTGF tumours (as shown in Figure 1a). Tracking the phagocytic capacity of these APC using fluorescent latex beads showed that TGF enhanced macrophage but decreased DC phagocytosis (Figure 3b). Hence, TGF not only alters the ratio of DC to macrophages but also directly influences their ability to phagocytose particles.

Figure 3.

TGF alters the APC ratio and enhances macrophage phagocytosis in vitro. (a) A total of 2 10⁶ whole spleen single-cell suspensions were incubated at various TGF concentrations for 24 h. CD11c⁺CD11b⁺ DC () and CD11c^-CD11b⁺ Macs () were identified by flow cytometry and the percentage of cells at the various TGF concentrations was determined. (b) Fluorescently labelled latex beads were added for the final 3 h of incubation and the level of bead uptake was determined by flow cytometry. The numbers shown in the histograms refer to the number of beads that had been phagocytosed by each cell. The total number of phagocytic DC () and Macs () was analysed by flow cytometry and the change induced by TGF is shown in the right panel. APC, antigen-presenting cell; DC, dendritic cells; TGF, transforming growth factor-₁.

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Macrophages are the dominant phagocytic cells infiltrating progressing skin tumours in vivo

Previously we have used fluorescent beads to successfully create a snapshot in time of the phagocytic capacity of tumour infiltrating APC.¹⁰ PGW- and PGWTGF tumour-bearing mice were injected intravenously with fluorescent beads 18 h prior to the removal and analysis of single cells by flow cytometry. A combination of anti-mouse CD11c, CD11b and MHC II was used to assess the phenotype of cells which had taken up the beads (Figure 4a). Similar to Figure 2a, DCs were identified as CD11c⁺CD11b⁺ while TAM as CD11c^-CD11b⁺. Moreover, consistent with the data presented in Table 1, compared to DC, TAM had a significantly reduced expression of MHC II (Figure 4a). Total number of bead⁺ cells was significantly greater in regressing PGW compared to progressing PGWTGF tumours (Figure 4b). More detailed analysis of the tumour infiltrating bead⁺ APC subsets revealed that only 12 and 3% of the total DC-infiltrating PGW and PGWTGF tumours respectively had phagocytosed beads (Figure 4c). In contrast, almost half of the TAM within progressing PGWTGF tumours were phagocytic within the time frame of the experiment (Figure 4c). Hence, TAM have a much greater phagocytic capacity than DC, and in progressing skin tumours, despite their relatively small number compared to DC (Figure 2b), they are the dominant phagocytic cells.

Figure 4.

Macrophages are the dominant phagocytic cells infiltrating progressing skin tumours. (a) Injected intravenously 18 h prior to tumour removal and analysis by four-colour flow cytometry. The phenotype of the cells that had taken up beads were confirmed as either CD11c⁺CD11b⁺MHC II^high DC (bold histogram) or CD11c^-CD11b⁺MHC II⁺ TAM (shaded histogram). Progressor tumour dot plots only are displayed as regressor dot plots looked similar. (b) Significantly more beads were taken up by any type of cell (percentage of total) within regressor (solid bar) compared to progressor (open bar) tumours (n=7). (c) Analysis of the percentage of each APC subset (DC in solid bars statistically compared to TAM in the open bar) that had taken up beads (n=7). In all cases mean+s.e.m. shown. ^*P<0.05. APC, antigen-presenting cell; DC, dendritic cells; TAM, tumour-associated macrophages.

Full figure and legend (154K)

Discussion

The presence of phagocytic macrophages within TGF-secreting progressing but not vector-transfected regressing tumour clones demonstrates the importance of these cells in orchestrating tumour growth. Transfection of a regressor skin tumour cell line with the gene for TGF enabled evasion of immunological destruction resulting in tumour progression in vivo. Because we have found previously that progression can be associated with an accumulation of macrophages inside the tumour,¹⁰ in a different model, we were interested in determining whether the TGF-mediated transformation of a regressor into a progressor tumour would also be associated with enhanced macrophage infiltration. Indeed, PGWTGF tumours were populated by significantly more macrophages and less DC than PGW regressor tumours indicating that TGF regulates the type of APC within skin tumours. Monocytes express high-affinity TGF type I/II receptors and TGF is a chemotactic factor for monocytes;^{15, 16} it is therefore possible that tumour-derived TGF acts as a chemokine recruiting peripheral blood monocytes to the tumour site where they differentiate into tissue-associated macrophages in preference to DC. The presence of high amounts of TGF would favour this macrophage pathway for two reasons, (1) monocytes that migrate through endothelium differentiate into DC, while those that remain in the subendothelial matrix become macrophages,¹⁷ and (2) TGF can disrupt cell junctions in endothelial layers.¹⁸ Hence, monocytes may be pushed to differentiate into macrophages rather than DC if TGF disruption of the endothelium allows monocytes to migrate into the tumour without crossing the endothelium. Our in vitro data showing that TGF alters the APC ratio by increasing the proportion of macrophages while at the same time decreasing DC percentages is novel and supports this hypothesis. Alternatively, the increase in TAM number following TGF transfection may be explained by local monocyte/macrophage proliferation,¹⁹ although whether macrophages are recruited to the tumour site, or undergo local proliferation is unclear.

In certain malignant diseases, accumulation of TAM within a tumour mass is associated with a poor prognostic outcome. The precise reasons for this is not clear, though the consensus is that TAM facilitate tumour immune evasion, progression and metastasis via a coordinated release of immunosuppressive, angiogenic and cellular growth factors (reviewed in Elgert et al.¹²). In addition, TAM have a high phagocytic capacity that enables them to efficiently phagocytose apoptotic tumour cells.^{20, 21} This is potentially an important immune evasion mechanism as the more efficient phagocytic TAM compete and prevent DC from taking up tumour antigens. Indeed, DC access to apoptotic tumour cells is a key event in the initiation of strong and sustained T-cell-mediated antitumour immunity.²² We used two approaches to address this issue. An in vitro assay of phagocytic capacity showed that consistent with previous reports,²³ TGF enhances macrophage uptake of fluorescent latex beads while concurrently inhibiting DC phagocytosis. This fluorescent latex bead-based assay was also used to measure the in vivo phagocytic capacity of APC within tumours and demonstrated that almost half of all the macrophages within progressor tumours were highly phagocytic. The advantages of this assay is that phagocytic cells can be tracked by flow cytometry. Unfortunately, this only provides a snapshot of the level of phagocytic activity inside a tumour at a given point in time. Despite this limitation, even after a relatively short period of time (18 h after bead transfer), we could still identify numerous phagocytic cells within the tumours. Based on these results, we hypothesize that one-way TGF-secreting tumours evade the immune system is through preferential recruitment of macrophages which in turn remove dead and dying tumour cells thereby preventing DC access to immunogenic tumour antigens.

Peripheral uptake of antigen by DC and their subsequent maturation and migration to draining lymph nodes is a key event in the development of immunity. TGF may subvert this process at a number of levels. TGF-exposed monocytes develop into immature DC that express high levels of E-cadherin and CCR6,²⁴ both of which are molecules that anchor DC to peripheral skin sites. This may explain how TGF-secreting progressor tumours evade immune detection by trapping DC at the tumour site² and preventing their migration to draining lymph nodes.³ However, even if DC from a TGF-secreting progressor tumour did successfully reach the draining lymph nodes, evidence from Kobie et al.²⁵ showing that TGF inhibits the antigen-presenting function of DC suggests that these APC may fail to activate tumour-specific T cells. Indeed, immature DC presenting antigen to naive T lymphocytes preferentially activates regulatory T-cell subsets.^{26, 27} Moreover, Sato et al.²⁸ showed that mature DC exposed to TGF failed to upregulate surface CCR7 and so were defective in their ability to migrate towards lymph node-derived CCL19. These multi-faceted, downregulating effects of TGF on the ability of DC to migrate and prime tumour-specific T cells may explain why TGF-secreting tumours grow progressively. However, based on our flow cytometric evidence, DC within both tumour groups had a similar phenotypic profile suggesting that DC in the tumour-draining lymph nodes dictate the outcome of the antitumour response. Based on this current study and our previous experiments showing a significant increase in skin-derived DC in regressor tumour-draining lymph nodes,³ we hypothesize that mature DC migrate away from regressor (but not progressor) tumours where they activate effective antitumour immune responses. In contrast, TGF secretion by tumours pushes DC precursors to differentiate into macrophages. Therefore, inhibition of TGF by aggressive tumours in the clinic may facilitate not only the migration of mature, antigen-primed DC to draining lymph nodes, but prevent the differentiation of DC precursors into immune suppressive tumour-associated macrophages. Ultimately it is hoped this will enhance the overall antitumour immune response.

Methods

Cell lines

Details of the tumour cell lines have been published by us previously.³ Briefly, a clone of the UV13.1 cell line was transfected with either an empty vector (PGW) or a vector containing the gene for TGF₁ (PGWTGF) by Transfectamine (Clontech, Palo Alto, CA, USA). The cell lines were maintained under routine tissue culture.

Tumour growth in mice

A total of 2 10⁶ PGW or PGWTGF tumour cells in 50 l phosphate-buffered saline were injected subcutaneously into the left and right flanks of female C3H/HeN (H-2^k) mice. All animals were aged 8–14 weeks and were used in accordance with animal ethics guidelines (University of Sydney, Australia). Tumour diameter was measured using an engineers' micrometer (Mitutoyo Corporation, Japan), taking the average of two diameter measurements. PGWTGF tumours grew progressively throughout the tumour monitoring, while PGW tumours grew progressively until around day 14 when they began to regress spontaneously. This was the time that the tumours were excised for analysis.

Measurement of TGF production by tumour cells in vitro

A total of 5 10⁶ tumour cells ml^-1 in a 24-well plate were cultured in Dulbecco's modified Eagle's medium with 2% fetal calf serum and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid for 24 h at 37 °C, 5% CO₂. Supernatant was collected and treated with 1 M HCl for 1 h at 4 °C and then neutralized with 1 M NaOH to activate any latent TGF. An enzyme-linked immunosorbent assay for TGF was subsequently performed on the cell culture supernatants with an OptEIA Set human TGF- kit (Pharmingen, San Diego, CA, USA).

Flow cytometric analysis of tumour-infiltrating antigen-presenting cells

The method of making single-cell suspensions from in vivo-grown solid tumours and the subsequent preparation of those cells for analysis by flow cytometry has been described by us previously.¹⁰ Single cells were incubated with antibodies against mouse CD45 (to identify cells of haematopoietic origin), CD11c (to identify DCs) and MHC Class II (to determine the maturation state of the antigen-presenting cells (APC)). Four-colour flow cytometry was then used to phenotype and enumerate the tumour-infiltrating leukocytes. CD45 (30-F11), CD11c (HL3), CD11b (Mac-1), I-A^k (11–5.2), CD80 (16–10A1) and CD86 (GL1) all from Pharmingen BD (Franklin Lakes, NJ, USA). Anti-mouse CCR6 (clone: 140706) was obtained from R&D Systems (Minneapolis, MN, USA). Murine CCR7 expression was detected using a fusion protein of human IgG fused to CCL19 (the specific chemokine for CCR7). The fusion protein was secreted by the cell line J558L (a kind gift from Jason G Cyster, Howard Hughes Medical Institute, University of California) and used directly from the culture supernatant generated in this laboratory as described previously.²⁹ Bound fusion protein was detected with phycoerytherin- conjugated anti-human IgG (Jackson Immunoresearch, West Grove, PA, USA). Isotype control antibody staining was used in parallel to ensure antibody specificity and to produce the electronic gates for analysis. Streptavidin allophycocyanate (Pharmingen) was routinely used to label biotinylated primary antibodies. Cellular events were acquired on a BD FACScalibur flow cytometer and analysed using CellQuest (Becton Dickinson, Franklin Lakes, NJ, USA) and FlowJo software (Treestar Inc., Ashland, OR, USA).

In vivo measurement of phagocytosis

Two hundred microliters of Fluoresbrite Carboxy YG or control non-fluorescent 2.6% solid latex microspheres with a diameter of 0.5 m (Polysciences; Warrington, PA, USA) were injected intravenously into tumour-bearing mice. Tumours were removed 18 h later and single-cell suspensions were incubated with 1 ml of 20 mM ethylenediaminetetraacetic acid (Sigma, St Louis, MO, USA) in phosphate-buffered saline for 20 min at room temperature prior to labeling with antibodies to ensure beads were intracellular.

Effect of TGF on macrophage and dendritic cell number and phagocytosis in vitro

A single-cell suspension was prepared from the spleen of a C3H/HeN mouse and 2 10⁶ cells at a concentration of 10⁶ ml^-1 were incubated in a 24-well plate with various indicated concentrations of recombinant hTGF (R&D Systems). Fluoresbrite Carboxy YG beads at a final concentration of 0.013% were added 21 h later and incubated for a further 3 h. At this time, suspended and adherent cells were recovered with an ethylenediaminetetraacetic acid/trypsin solution and washed extensively with ethylenediaminetetraacetic acid to remove surface beads. DC and macrophages were identified by flow cytometry using antibodies against CD11c and CD11b.

Statistical analysis

An unpaired two-tailed Student's t-test was used to analyse TGF production for each clone by enzyme-linked immunosorbent assay. In vivo tumour growth was analysed using repeated measures of analysis of variance. Tumour incidence was analysed using Kaplan–Meier Survival test. Flow cytometry data and bead uptake experiments were all analysed using a post hoc Fisher's protected least significant difference (PLSD) analysis of variance test. For all analyses, a P-value of <0.05 was considered significant.

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Acknowledgements

This work was supported by a grant from the Cure Cancer Australia Foundation. SNB is supported by an NH&MRC CJ Martin Fellowship (no. 307726).