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Gut Online First, published on July 8, 2015 as 10.1136/gutjnl-2015-309193
Pancreas
ORIGINAL ARTICLE
Dual prognostic significance of tumour-associated macrophages in human pancreatic adenocarcinoma treated or untreated with chemotherapy Giuseppe Di Caro,1 Nina Cortese,1 Giovanni Francesco Castino,1 Fabio Grizzi,1 Francesca Gavazzi,2 Cristina Ridolfi,2 Giovanni Capretti,2 Rossana Mineri,3 Jelena Todoric,4 Alessandro Zerbi,2 Paola Allavena,1 Alberto Mantovani,1 Federica Marchesi1 ▸ Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ gutjnl-2015-309193). For numbered affiliations see end of article. Correspondence to Dr Federica Marchesi, Department of Immunology and Inflammation, Humanitas Clinical and Research Center, Via Manzoni 56, Rozzano (MI) 20089, Italy; federica.marchesi@ humanitasresearch.it GDC and NC contributed equally. Received 14 January 2015 Revised 15 June 2015 Accepted 21 June 2015
ABSTRACT Objective Tumour-associated macrophages (TAMs) play key roles in tumour progression. Recent evidence suggests that TAMs critically modulate the efficacy of anticancer therapies, raising the prospect of their targeting in human cancer. Design In a large retrospective cohort study involving 110 patients with pancreatic ductal adenocarcinoma (PDAC), we assessed the density of CD68-TAM immune reactive area (%IRA) at the tumour–stroma interface and addressed their prognostic relevance in relation to postsurgical adjuvant chemotherapy (CTX). In vitro, we dissected the synergism of CTX and TAMs. Results In human PDAC, TAMs predominantly exhibited an immunoregulatory profile, characterised by expression of scavenger receptors (CD206, CD163) and production of interleukin 10 (IL-10). Surprisingly, while the density of TAMs associated to worse prognosis and distant metastasis, CTX restrained their protumour prognostic significance. High density of TAMs at the tumour–stroma interface positively dictated prognostic responsiveness to CTX independently of T-cell density. Accordingly, in vitro, gemcitabine-treated macrophages became tumoricidal, activating a cytotoxic gene expression programme, inhibiting their protumoural effect and switching to an antitumour phenotype. In patients with human PDAC, neoadjuvant CTX was associated to a decreased density of CD206+ and IL-10+ TAMs at the tumour–stroma interface. Conclusions Overall, our data highlight TAMs as critical determinants of prognostic responsiveness to CTX and provide clinical and in vitro evidence that CTX overall directly re-educates TAMs to restrain tumour progression. These results suggest that the quantification of TAMs could be exploited to select patients more likely to respond to CTX and provide the basis for novel strategies aimed at re-educating macrophages in the context of CTX.
Significance of this study What is already known on this subject?
▸ Tumour-associated macrophages (TAMs) are an important component of the microenvironment of solid tumours, in the majority of cancers associated to unfavourable prognosis. However, their peculiar plasticity allows them to acquire either protumour or antitumour functions. Therefore, TAMs emerge as attractive targets of therapeutic strategies aimed at reprogramming their protumour phenotype into an effective antitumour activity. ▸ Human pancreatic ductal adenocarcinoma (PDAC) is a very aggressive disease, displaying low responsiveness to conventional therapies and characterised by an immunosuppressive microenvironment.
What are the new findings?
▸ In human PDAC, macrophages accumulate at the tumour–stroma interface and display an immunosuppressive phenotype. However, density of macrophages is a critical determinant in identifying patient responsiveness to conventional chemotherapy (CTX). ▸ In vitro, CTX synergises with TAM-mediated tumour cytotoxicity, preventing their tumour-protective role and reinstating their antitumour function, by a T-cell independent mechanism. ▸ In patients with PDAC, administration of neoadjuvant CTX is associated to a decreased density of protumour TAMs at the tumour– stroma interface, supporting the ability of CTX to modulate the function of TAMs in the tumour microenvironment.
INTRODUCTION To cite: Di Caro G, Cortese N, Castino GF, et al. Gut Published Online First: [please include Day Month Year] doi:10.1136/gutjnl2015-309193
Tumour-associated macrophages (TAMs) are a major component of the tumour immune microenvironment, involved in the majority of cancers in the orchestration of key steps required for cancer occurrence and progression.1–4 Even though this simplified view is substantiated by epidemiological
and experimental evidence,5–8 macrophages display a peculiar plasticity, which allows them to exert antagonistic functions, as they can acquire either protumour or antitumour functions;9 10 this makes TAMs attractive targets of therapeutic strategies
Di Caro G, et al. Gut 2015;0:1–11. doi:10.1136/gutjnl-2015-309193
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Copyright Article author (or their employer) 2015. Produced by BMJ Publishing Group Ltd (& BSG) under licence.
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Pancreas Significance of this study How might it impact on clinical practice in the foreseeable future?
▸ The small therapeutic window of CTX in patients with PDAC would greatly benefit by the identification of novel biomarkers with the ability to detect patients who are more likely to benefit from CTX. ▸ Despite its clinically aggressive behaviour, in human PDAC, targeting TAMs may be beneficial to tumour prognosis and in some cases be sufficient to ignite an effective antitumour action. ▸ New strategies enhancing CTX-driven antitumour activity might open the way for personalised medicine and more tailored therapeutic treatments.
aimed at reprogramming their protumour phenotype into an effective antitumour activity. The antitumour role of macrophages has been previously linked to the orchestration of T-cell antitumour immunity;11 however, recent results showed that tumour immune surveillance could be firmly directed by TAMs when educated by specific treatments, in a T-independent fashion.12 In this scenario, macrophages are being rediscovered as key regulators of tumour response to anticancer therapies.3 13–18 Pancreatic ductal adenocarcinoma (PDAC) is the fifth cause of cancer worldwide.19 Approximately 80% of cases are not eligible for surgery for either local or metastatic disease and have dismal survival rates at 1 year from diagnosis,20 with surgery being the only treatment with curative intent for patients with early diagnosis. Lately, new multidisciplinary perioperative approaches and the concentration of care in high volume centres have improved the prognosis of patients undergoing surgery.21 22 In this new scenario, the characterisation of PDAC cases with long survival could reveal novel biological determinants of progression for this disease, by comparison with patients with dismal prognosis. To date, most of the data generated by global analyses and standardised assessment of immune infiltrating cells have been focused on the prognostic determination of tumour-infiltrating lymphocytes (TILs) and suggested that the role of adaptive immune responses in controlling tumour growth is a common feature to most solid tumours.23 Notably, while TILs are candidate biomarkers in determining the prognosis in several cancers,23 the idea that TILs might be useful in identifying patients more responsive to conventional chemotherapies in humans23 is merely speculative.24 New strategies or new cellular mediators capable of enhancing chemotherapy (CTX)-driven antitumour activity are required to open the way for personalised medicine and more tailored therapeutic treatments. The small therapeutic window of CTX in patients with PDAC25–27 would greatly benefit from the identification of novel biomarkers with the ability to detect patients with PDAC who are more likely to benefit from chemoimmunotherapeutic approaches and would be highly beneficial to the cure of this disease. In this study, we approached the microenvironment of human pancreatic cancer by systematically evaluating the macrophage immune component at the tumour–host interface. The occurrence and the extent of CD68+ TAMs (CD68-TAMs) at the tumour–stroma interface were evaluated in a large consecutive 2
series of 110 patients with human pancreatic adenocarcinoma who underwent curative surgical resection. The clinical relevance of TAMs and whether their density associates to disease recurrence and metastatic potential in human PDAC were assessed in relation to postsurgical adjuvant CTX. Here, we show that the density of macrophages is a critical determinant in identifying patient responsiveness to conventional CTX and its response rate was highly modified by the presence of macrophages at the tumour–stroma interface. In in vitro analyses we dissected the synergism of CTX and TAMs and found that macrophages exposed to CTX switched to a proinflammatory phenotype and became tumoricidal by a TAM-autonomous and T-cell independent mechanism.
MATERIALS AND METHODS Patients and study design We designed a consecutive and retrospective observational cohort study including 110 patients aged older than 18 years, diagnosed with PDAC and who consecutively underwent surgery with curative intent in Humanitas Clinical and Research Center, from February 2010 to December 2012. All patients gave their informed consent prior to inclusion in the study, and the Ethics Committee of the Humanitas Clinical and Research Center approved the study. Gemcitabine (GEM)-based, postsurgical adjuvant CTX was allocated to most patients (see online supplementary table S1) by a non-random assignment according to adjuvant protocols in use at the time of surgery. Sixteen patients received a GEM-based neoadjuvant CTX regimen before surgery. The continuous, median and quartile values of the percentages of CD8-TIL and CD68-TAM IRA (immunoreactive area) were compared with demographics, clinical and histopathological features and all these variables were tested as predictors of patient’s outcome (disease-free survival, DFS), patient’s disease-specific survival (DSS) and metachronous metastasis. Further information is listed in the online supplementary files.
Immunohistochemistry and image analysis From each patient enrolled in the study, 2 mm thick tissue slides from formalin-processed and paraffin-embedded tumour sections were retrieved from the archive of the pathology department and processed for immunohistochemistry as previously done.28 Details on immunostaining and image analysis are available in the online supplementary files.
Macrophage cytotoxicity assays To assess macrophage cytotoxicity, coculture experiments of human macrophages and pancreatic cancer cells were performed in the presence or absence of GEM. Details are listed in the online supplementary files.
RESULTS Macrophages accumulate at the tumour–stroma interface in human PDAC and display an immunosuppressive phenotype To evaluate the contribution of TAMs to the immune microenvironment of human PDAC, we systematically and quantitatively assessed by immunohistochemistry the percentage of IRA of CD68+ macrophages (CD68-TAM %IRA) at the tumour–stroma interface (figure 1A and see online supplementary table S1), in 110 tissue specimens from patients with PDAC who underwent surgical resection at our institution. Whole tissue slide quantification evidenced that the density of CD68-TAMs was heterogeneous among patients with PDAC, with some cases showing a high infiltration (figure 1B) and others low infiltration Di Caro G, et al. Gut 2015;0:1–11. doi:10.1136/gutjnl-2015-309193
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Pancreas
Figure 1 CD68-TAMs at the tumour–stroma interface display an immunosuppressive phenotype. (A–D) Histological sections of human PDAC, stained for CD68-TAMs. The image exemplifies the methodology used for the assessment of CD68-TAMs at the tumour–stroma interface (dot line); cancer tissue represents approximately 50% of the microscopic field (A). The density of CD68-TAM cells differs greatly, with some highly infiltrated cases (B) and some scarcely infiltrated cases (C). Distribution of TAM density across 110 patients with PDAC (D). (E–I) TAMs display an immunosuppressive phenotype in human PDAC. Immunohistochemical stainings for CD68 (E), CD163 (F) and CD206 (G) on the same PDAC case and double staining with CD68 and IL-10 (H) antibodies show that TAMs in human PDAC predominantly exhibit an immunoregulatory phenotype. Arrowheads indicate CD68-TAMs expressing IL-10. The majority of macrophages express CD206 (62.4±16.9%, n=12, p=0.003), while IL-12 is expressed only by a minority of macrophages (8.7±10.1%, n=6, p=0.0006). ( J–N) CD68-TAMs in human PDAC localise in regions dominated by an immunosuppressive milieu. TAMs ( J) surrounding a tumour gland expressing the chemokine CCL17 (K), but not CXCL10 (L). CD68-TAMs (M) were also a source of CCL17 (N), associated to a Th2-type response. Arrowheads indicate CD68-TAMs expressing CCL17. Sections in E–G and in J–L and M and N are consecutive sections. Bars: (A) 500 μm, (B and C) 200 μm, ( J–L) 100 μm, (H, M and N) 10 μm. **p=0.003; ***p=0.0006, by Student’s t test. IL, interleukin; %IRA, percentage immunoreactive area; PDAC, pancreatic ductal adenocarcinoma; TAMs, tumour-associated macrophages.
(figure 1C) at the tumour–stroma interface. Figure 1D reports distribution of TAM density across 110 patients with PDAC. Macrophages may have a spectrum of different polarisation states, which dictate their function in the tumour context.10 Accordingly, macrophages at the tumour–stroma interface of pancreatic cancer (figure 1E) predominantly exhibited an immunosuppressive phenotype, as shown by high expression of receptors CD163 (figure 1F), CD206 (figure 1G) and interleukin-10 (IL-10), a prototypical immunosuppressive cytokine (figure 1H). Notably, while expression of CD206 identified most of the CD68+ macrophages present at the tumour–stroma interface (mean%±SD of positive cells 62.4±16.9, n=12, p=0.003), IL-12, which is mainly associated to an M1-like polarisation state in vitro, was expressed only by a minority of macrophages (mean%±SD of positive cells 8.7±10.1, n=6, p=0.0006) (figure 1I), confirming the consolidated evidence that TAMs in human PDAC are prevalently immunosuppressive.11 29 CD68-TAMs (figure 1J) frequently localised in the proximity of Di Caro G, et al. Gut 2015;0:1–11. doi:10.1136/gutjnl-2015-309193
tumour glands expressing the chemokine CCL17 (figure 1K), a Th2-type chemokine, and low or any amounts of CXCL10 (figure 1L), an exemplar interferon (IFN)-γ-induced chemokine, suggesting that CD68-TAMs in human PDAC localise in regions dominated by an immunosuppressive milieu. Notably, CD68-TAMs (figure 1M) were also a source of CCL17 themselves (figure 1N), which is associated to a Th2-type response.
Density of CD68-TAMs at the tumour–stroma interface predicts response to CTX We next analysed the distribution at the tumour–stroma interface and the prognostic value of CD68-TAMs in the microenvironment of human PDAC. In online supplementary table S2, univariate and multivariate Cox regression analyses are shown including demographics, clinical and histopathological features and the density of CD68-TAMs. We recorded 72 (65.7%) outcome events of DFS among 110 patients with PDAC. At multivariate Cox analysis, TNM staging and tumour grade were 3
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Pancreas Table 1 CD68-TAMs as predictors of postsurgical disease-specific recurrences in 110 patients with pancreatic ductal adenocarcinoma, according to CTX treatment Adjuvant CTX treatment No Recurrence rate (%) CD68-TAMs (%IRA)† (quartiles) First 4/12 (33) Second 5/7 (71) Third 4/9 (44) Fourth 7/8 (88)
Yes HR (95% CI)*
p Value
Recurrence rate (%)
HR (95% CI)*
p Value
1.00 2.40 2.29 4.43
0.20 0.26 0.02
13/16 14/20 14/18 11/20
1.00 0.72 0.70 0.37
0.39 0.35 0.01
ref. (0.64 to 8.97) (0.44 to 7.50) (1.21 to 16.24)
(81) (70) (78) (55)
ref. (0.34 to 1.54) (0.33 to 1.50) (0.16 to 0.83)
*Cox regression analysis. †Densities as percentage immunoreactive area at the tumour invasive front. CTX, chemotherapy; TAMs, tumour-associated macrophages.
significantly associated with higher risk of disease recurrence (see online supplementary table S2). Notably, by systematically analysing all the variables that may interact in determining patient prognosis, including the densities of immune cell populations, we found a significant interaction between the density of CD68-TAMs and adjuvant CTX ( p=0.004) (see online supplementary table S2). To assess whether CTX interacted also with the adaptive immune infiltrate, we also evaluated the density of CD8-TILs (CD8-TIL %IRA) (see online supplementary figure S1A and tables S1 and S2). In contrast to CD68-TAMs, densities of CD8-TILs did not show interaction with CTX in predicting the risk of patient’s outcome ( p=0.84) (see online supplementary table S2). Pearson’s linear regression analysis revealed no association between distributions of CD68-TAM %IRA and CD8-TILs %IRA (r=0.07, p=0.49) (see online supplementary figure S1B), suggesting that these immune populations belong to distinct immune networks in the tumour microenvironment. To dissect the interaction of CTX and CD68-TAMs, we designed a subgroup analysis and investigated the prognostic value of CD68-TAMs in patients with PDAC according to the administration of postsurgical CTX (table 1). Surprisingly, while among patients who did not receive any adjuvant CTX (n=36), cumulative increasing values of CD68-TAMs were associated with a higher risk of disease progression (HR=1.57; 95% CI (1.04 to 2.37); p=0.03), among patients with PDAC receiving postsurgical adjuvant CTX (n=74), increasing values of CD68-TAM densities were associated with a better prognosis (HR=0.74; 95% CI (0.58 to 0.95); p=0.02)) (see online supplementary table S1 and figure S2). This result indicates that the prognostic value of TAMs in predicting tumour recurrence is critically influenced by postsurgical
CTX. This evidence was also confirmed by analysing whether response to adjuvant CTX varied according to the density of TAMs at the tumour–stroma interface (table 2). Interestingly, among patients with high densities of CD68-TAMs (fourth quartile, n=28) CTX associated with lower risk of disease recurrence (n=28; HR: 0.38; 95% CI (0.13 to 1.00); p=0.05). Differently, CTX associated with worst prognosis in patients with PDAC with low CD68-TAMs (first to third quartile, n=82) (n=82; HR: 2.19; 95% CI (1.15 to 4.16); p=0.02). This suggests that the presence of CD68-TAMs at the tumour–stroma interface highly influences responsiveness of patients with PDAC to CTX, and could therefore be required for CTX efficacy. Therefore, CD68-TAM assessment could be exploited to identify a subgroup of patients with PDAC highly responsive to CTX and who could receive prognostic advantage from this therapeutic adjuvant treatment. In light of the immunosuppressive phenotype of macrophages we described in PDAC, we tested in our human cohort whether the amount of TAMs at the tumour–stroma interface modulates the prognostic abilities of CD8-TILs after CTX. We found that among patients treated with CTX, the density of CD8-TILs was not a prognostic variable in both patients with low (median) CD68-TAMs (p=0.94, p=0.46) (see online supplementary figure S1C). This result suggests that TAMs are independent of TILs in predicting prognosis and thus their antitumour activities seems to be mediated by T-cell independent pathways. TAMs are known to greatly influence the occurrence of distant metastases.6 30–32 The interaction with postsurgical CTX in patients with PDAC suggests that they might have a role in the suppression of micrometastases, occurring after surgical resection. To investigate this hypothesis, we evaluated the association of TAM density to distant metachronous metastases.
Table 2 Adjuvant CTX treatment as predictor of postsurgical disease-specific recurrences in 110 patients with PDAC, according to the extent of CD68-TAMs at the tumour–stroma interface CD68-TAM (%IRA)* Low (first to third quartile) Recurrence rate (%) Adjuvant chemotherapy treatment No 13/28 (46) Yes 41/54 (76)
High (fourth quartile) HR (95% CI)†
p Value
Recurrence rate (%)
HR (95% CI)†
p Value
2.19 (1.15 to 4.16)
0.02
7/8 (88) 11/20 (55)
0.38 (0.13 to 1.00)
0.05
*Densities as %IRA at the tumour invasive front. †Cox regression analysis. CTX, chemotherapy; %IRA, percentage immunoreactive area; PDAC, pancreatic ductal adenocarcinoma; TAMs, tumour-associated macrophages.
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Pancreas Table 3 CD68-TAMs as predictors of postsurgical distant organ metachronous metastasis in 110 patients with PDAC, according to adjuvant chemotherapy treatment Adjuvant chemotherapy treatment No Metachronous metastasis (%) CD68-TAM (%IRA)† (quartiles) First 3/12 (25) Second 2/7 (29) Third 4/9 (44) Fourth 6/8 (75)
Yes HR (95% CI)*
p Value
Metachronous metastasis (%)
HR (95% CI)*
p Value
1.00 ref. 1.22 (0.20 to 7.34) 2.94 (0.62 to 13.94) 5.10 (1.18 to 22.11)
0.83 0.17 0.02
7/16 10/20 11/18 11/20
1.00 1.01 1.05 0.76
0.97 0.92 0.58
(44) (50) (61) (55)
ref. (0.39 to 2.67) (0.40 to 2.73) (0.29 to 1.99)
*Cox regression analysis. †Densities as percentage immunoreactive area (IRA) at the tumour invasive front. %IRA, percentage immunoreactive area; PDAC, pancreatic ductal adenocarcinoma; TAMs, tumour-associated macrophages.
Significantly, among patients with PDAC who did not receive CTX (n=36), cumulative increasing densities of CD68-TAMs were significantly associated with higher risk of metachronous metastasis (HR=1.77; 95% CI (1.09 to 2.87); p=0.02), while among those who received CTX (n=74) the densities of CD68-TAMs were not associated with prognosis (HR=0.91; 95% CI (0.69 to 1.22); p=0.54) (table 3, see online supplementary figure S2). This result suggests that CD68-TAMs could promote distant metastasis occurrence and that CTX could restrain their prometastatic activity, as evidenced also by Kaplan–Meier prognostic curves (figure 2A, B, see online supplementary figure S2). On the contrary, when we investigated patient’s DSS, we found that TAMs were significantly associated with better survival only among patients who received adjuvant CTX (p=0.05) (figure 2D), while among those untreated, TAM had a tendency to associate to worst prognosis, although not significant (p=0.12) (figure 2C). These results highlight a synergism between CTX and TAMs that differs according to the prognostic clinical endpoint and this could reflect the different biological roles of TAMs in local versus metastatic distant tumour progression.
GEM synergises with TAM-mediated tumour cytotoxicity To provide insight into the interaction between TAMs and CTX, we performed in vitro experiments of tumour cytotoxicity in the presence of differently polarised macrophages and GEM. At an early time point (24 h), treatment of the PDAC cell line (MiaPaCa-2) with 500 mM GEM resulted in a slightly enhanced but not significant tumour cell death (figure 3A, left); however at later time points (48–72 h), the treatment was toxic on tumour cells, confirming that the latter were susceptible to the drug (not shown). M1 macrophages polarised with INF-γ and lipopolysaccharide (LPS) alone did not induce significant increase in PDAC cell cytotoxicity (figure 3A, middle), consistent with the ability of PDAC cells to dampen macrophage antitumour function. By contrast, when GEM was added to the coculture, there was a significant increase in tumour cell death, suggesting that M1 macrophages and CTX have a synergic cytotoxic effect (figure 3A, middle and 3B, left). In contrast, there was a significant decrease in tumour cell death when PDAC tumour cells were cocultured with M2-like macrophages ( polarised with IL-4) compared with PDAC cells alone, suggesting that M2-like macrophages exhibit a protective role towards PDAC tumour cells in vitro ( p≤0.001) (figure 3A, right). However, surprisingly, GEM treatment was able to significantly revert the protective effect of M2-like macrophages (figure 3A, Di Caro G, et al. Gut 2015;0:1–11. doi:10.1136/gutjnl-2015-309193
right and 3B, right panel), reinstating its original cytotoxic effect ( p≤0.01). A dose–response curve with increasing concentrations of GEM showed the specificity of the synergic effect and confirmed that this effect was already induced at low drug concentrations (figure 3C), while at the highest concentration it was not further enhanced. Collectively, these findings reveal that GEM modulates the interaction of macrophages with PDAC cells in vitro and reflect the interaction of GEM and macrophages observed in our human study. We then attempted to dissect the synergism of GEM and macrophages. GEM treatment induced neither selective death of immunosuppressive macrophages (figure 4A) nor tumour cell release of molecules able to activate or reprogram macrophages (figure 4B, C). In contrast, tumour cell death was significantly dose-dependently increased when PDAC tumour cells were cocultured with macrophages pretreated with GEM (MfpreGEM), compared with those cocultured with control macrophages (figure 4B, D), thus suggesting that GEM exerted its modulatory effect by acting directly on the macrophages and thwarting the protective function of macrophages towards PDAC cells. Notably, depending on the cell line tested, the interaction between GEM and macrophages was either abrogated (figure 4D, left panel) or maximum (figure 4D, right panel) at the highest GEM dose. We then investigated the mechanism whereby GEM enhanced macrophage cytotoxicity. Inhibition of macrophage death signals, including tumour necrosis factor (TNF)-α and TRAIL, did not affect the synergism between macrophages and GEM in killing PDAC tumour cells (see online supplementary figure S3). In contrast, GEM treatment induced an early and dramatic increase in cellular reactive oxygen species (ROS) in macrophages (figure 4E), thus suggesting that release of cytotoxic ROS could be responsible for the induced macrophage antitumour activity.
GEM directly modifies macrophage polarisation in vitro and in the tumour microenvironment of human PDAC To reveal molecular actors regulating the CTX-mediated modulation of macrophage function, we evaluated the effect of GEM on the mRNA expression profile of macrophages. GEM-treated macrophages significantly increased expression of several immune-stimulating cytokines, such as IL-12, IL-27, IFNG, as well as cytokines typically involved in inflammatory responses, including prostaglandin-endoperoxide synthase, Fas ligand and TNF-α (figure 5A). In accordance with the evidence that GEM induces ROS production in macrophages (figure 4E), there was also an increase in molecules regulating microbial responses such as inducible nitric oxide synthase and nicotinamide 5
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Pancreas
Figure 2 Clinical relevance of CD68-TAMs in human pancreatic adenocarcinoma. (A and B) Kaplan–Meier curves showing metachronous metastasis, according to CD68-TAM density and CTX. A high density (≥median) of CD68-TAMs associated to an increased risk of distant tumour metastasis compared with a lower density only in patients who did not receive adjuvant CTX ( p=0.02, n=36) (A), but not in those CTX treated ( p=0.68, n=74) (B). (C and D) Kaplan–Meier curves showing DSS%, according to CD68-TAM density and CTX. A high density (≥median) of CD68-TAMs was significantly associated with better survival only among patients who received adjuvant CTX ( p=0.05) (C), while there was a tendency to associate to worst prognosis, but not significant in untreated patients ( p=0.12) (D). CTX, chemotherapy; DSS, disease-specific survival; TAMs, tumour-associated macrophages.
adenine dinucleotide phosphate, although not significant. Remarkably, the increase in proinflammatory mediators was paralleled by a significant decrease in IL-10, also confirmed by decreased protein production (see online supplementary figure S5), suggesting an overall switch in activation status. In accordance with these results, the gene expression profile of GEM-treated macrophages also evidenced a significantly decreased expression of some growth factors, such as platelet growth factor A and B, placental growth factor and matrix proteins, such as osteopontin and fibronectin (figure 5A), which are mediators typically upregulated in protumour M2-like TAM. Overall, these findings strongly suggest that GEM treatment acts directly on macrophages by inducing a downregulation of trophic factors, concurrently inducing an increased expression of inflammatory mediators able to revert this protective function and likely to explain the protumour activity of CTX-untreated macrophages. In accordance with gene expression data, GEM treatment significantly modified surface expression of polarising markers, upregulating expression of the M1-markers HLA-DR, CD40 and the chemokine receptor CCR7 and downregulating the M2-markers CD163 and CD206 (figure 5B, C). To further confirm the effect of CTX on macrophage repolarisation, we took advantage of a cohort of consecutive patients who were administered neoadjuvant CTX and evaluated the phenotype of macrophages at the tumour front, by immunohistochemistry on tumour tissue slides. The density of CD68-TAMs and CD8-TILs at the tumour–stroma interface was not significantly modified after neoadjuvant CTX, although a slight tendency to decreased density of CD68-TAMs was 6
observed ( p=0.11, see online supplementary table S1). However, despite a comparable density, the phenotype of macrophages at the tumour–stroma interface was affected by CTX administration: while in untreated patients a considerable amount of CD68-TAMs expressed the M2-marker CD206 (figure 5D, upper panel), significantly less macrophages were CD206-positive in PDAC sections from neoadjuvant-treated patients (figure 5D, lower panel and 5E). Similarly, while the majority of macrophages in human PDAC was IL-10+, in patients treated with CTX the presence of IL-10+ macrophages decreased significantly ( p
Gut Online First, published on July 8, 2015 as 10.1136/gutjnl-2015-309193
Pancreas
ORIGINAL ARTICLE
Dual prognostic significance of tumour-associated macrophages in human pancreatic adenocarcinoma treated or untreated with chemotherapy Giuseppe Di Caro,1 Nina Cortese,1 Giovanni Francesco Castino,1 Fabio Grizzi,1 Francesca Gavazzi,2 Cristina Ridolfi,2 Giovanni Capretti,2 Rossana Mineri,3 Jelena Todoric,4 Alessandro Zerbi,2 Paola Allavena,1 Alberto Mantovani,1 Federica Marchesi1 ▸ Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ gutjnl-2015-309193). For numbered affiliations see end of article. Correspondence to Dr Federica Marchesi, Department of Immunology and Inflammation, Humanitas Clinical and Research Center, Via Manzoni 56, Rozzano (MI) 20089, Italy; federica.marchesi@ humanitasresearch.it GDC and NC contributed equally. Received 14 January 2015 Revised 15 June 2015 Accepted 21 June 2015
ABSTRACT Objective Tumour-associated macrophages (TAMs) play key roles in tumour progression. Recent evidence suggests that TAMs critically modulate the efficacy of anticancer therapies, raising the prospect of their targeting in human cancer. Design In a large retrospective cohort study involving 110 patients with pancreatic ductal adenocarcinoma (PDAC), we assessed the density of CD68-TAM immune reactive area (%IRA) at the tumour–stroma interface and addressed their prognostic relevance in relation to postsurgical adjuvant chemotherapy (CTX). In vitro, we dissected the synergism of CTX and TAMs. Results In human PDAC, TAMs predominantly exhibited an immunoregulatory profile, characterised by expression of scavenger receptors (CD206, CD163) and production of interleukin 10 (IL-10). Surprisingly, while the density of TAMs associated to worse prognosis and distant metastasis, CTX restrained their protumour prognostic significance. High density of TAMs at the tumour–stroma interface positively dictated prognostic responsiveness to CTX independently of T-cell density. Accordingly, in vitro, gemcitabine-treated macrophages became tumoricidal, activating a cytotoxic gene expression programme, inhibiting their protumoural effect and switching to an antitumour phenotype. In patients with human PDAC, neoadjuvant CTX was associated to a decreased density of CD206+ and IL-10+ TAMs at the tumour–stroma interface. Conclusions Overall, our data highlight TAMs as critical determinants of prognostic responsiveness to CTX and provide clinical and in vitro evidence that CTX overall directly re-educates TAMs to restrain tumour progression. These results suggest that the quantification of TAMs could be exploited to select patients more likely to respond to CTX and provide the basis for novel strategies aimed at re-educating macrophages in the context of CTX.
Significance of this study What is already known on this subject?
▸ Tumour-associated macrophages (TAMs) are an important component of the microenvironment of solid tumours, in the majority of cancers associated to unfavourable prognosis. However, their peculiar plasticity allows them to acquire either protumour or antitumour functions. Therefore, TAMs emerge as attractive targets of therapeutic strategies aimed at reprogramming their protumour phenotype into an effective antitumour activity. ▸ Human pancreatic ductal adenocarcinoma (PDAC) is a very aggressive disease, displaying low responsiveness to conventional therapies and characterised by an immunosuppressive microenvironment.
What are the new findings?
▸ In human PDAC, macrophages accumulate at the tumour–stroma interface and display an immunosuppressive phenotype. However, density of macrophages is a critical determinant in identifying patient responsiveness to conventional chemotherapy (CTX). ▸ In vitro, CTX synergises with TAM-mediated tumour cytotoxicity, preventing their tumour-protective role and reinstating their antitumour function, by a T-cell independent mechanism. ▸ In patients with PDAC, administration of neoadjuvant CTX is associated to a decreased density of protumour TAMs at the tumour– stroma interface, supporting the ability of CTX to modulate the function of TAMs in the tumour microenvironment.
INTRODUCTION To cite: Di Caro G, Cortese N, Castino GF, et al. Gut Published Online First: [please include Day Month Year] doi:10.1136/gutjnl2015-309193
Tumour-associated macrophages (TAMs) are a major component of the tumour immune microenvironment, involved in the majority of cancers in the orchestration of key steps required for cancer occurrence and progression.1–4 Even though this simplified view is substantiated by epidemiological
and experimental evidence,5–8 macrophages display a peculiar plasticity, which allows them to exert antagonistic functions, as they can acquire either protumour or antitumour functions;9 10 this makes TAMs attractive targets of therapeutic strategies
Di Caro G, et al. Gut 2015;0:1–11. doi:10.1136/gutjnl-2015-309193
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Pancreas Significance of this study How might it impact on clinical practice in the foreseeable future?
▸ The small therapeutic window of CTX in patients with PDAC would greatly benefit by the identification of novel biomarkers with the ability to detect patients who are more likely to benefit from CTX. ▸ Despite its clinically aggressive behaviour, in human PDAC, targeting TAMs may be beneficial to tumour prognosis and in some cases be sufficient to ignite an effective antitumour action. ▸ New strategies enhancing CTX-driven antitumour activity might open the way for personalised medicine and more tailored therapeutic treatments.
aimed at reprogramming their protumour phenotype into an effective antitumour activity. The antitumour role of macrophages has been previously linked to the orchestration of T-cell antitumour immunity;11 however, recent results showed that tumour immune surveillance could be firmly directed by TAMs when educated by specific treatments, in a T-independent fashion.12 In this scenario, macrophages are being rediscovered as key regulators of tumour response to anticancer therapies.3 13–18 Pancreatic ductal adenocarcinoma (PDAC) is the fifth cause of cancer worldwide.19 Approximately 80% of cases are not eligible for surgery for either local or metastatic disease and have dismal survival rates at 1 year from diagnosis,20 with surgery being the only treatment with curative intent for patients with early diagnosis. Lately, new multidisciplinary perioperative approaches and the concentration of care in high volume centres have improved the prognosis of patients undergoing surgery.21 22 In this new scenario, the characterisation of PDAC cases with long survival could reveal novel biological determinants of progression for this disease, by comparison with patients with dismal prognosis. To date, most of the data generated by global analyses and standardised assessment of immune infiltrating cells have been focused on the prognostic determination of tumour-infiltrating lymphocytes (TILs) and suggested that the role of adaptive immune responses in controlling tumour growth is a common feature to most solid tumours.23 Notably, while TILs are candidate biomarkers in determining the prognosis in several cancers,23 the idea that TILs might be useful in identifying patients more responsive to conventional chemotherapies in humans23 is merely speculative.24 New strategies or new cellular mediators capable of enhancing chemotherapy (CTX)-driven antitumour activity are required to open the way for personalised medicine and more tailored therapeutic treatments. The small therapeutic window of CTX in patients with PDAC25–27 would greatly benefit from the identification of novel biomarkers with the ability to detect patients with PDAC who are more likely to benefit from chemoimmunotherapeutic approaches and would be highly beneficial to the cure of this disease. In this study, we approached the microenvironment of human pancreatic cancer by systematically evaluating the macrophage immune component at the tumour–host interface. The occurrence and the extent of CD68+ TAMs (CD68-TAMs) at the tumour–stroma interface were evaluated in a large consecutive 2
series of 110 patients with human pancreatic adenocarcinoma who underwent curative surgical resection. The clinical relevance of TAMs and whether their density associates to disease recurrence and metastatic potential in human PDAC were assessed in relation to postsurgical adjuvant CTX. Here, we show that the density of macrophages is a critical determinant in identifying patient responsiveness to conventional CTX and its response rate was highly modified by the presence of macrophages at the tumour–stroma interface. In in vitro analyses we dissected the synergism of CTX and TAMs and found that macrophages exposed to CTX switched to a proinflammatory phenotype and became tumoricidal by a TAM-autonomous and T-cell independent mechanism.
MATERIALS AND METHODS Patients and study design We designed a consecutive and retrospective observational cohort study including 110 patients aged older than 18 years, diagnosed with PDAC and who consecutively underwent surgery with curative intent in Humanitas Clinical and Research Center, from February 2010 to December 2012. All patients gave their informed consent prior to inclusion in the study, and the Ethics Committee of the Humanitas Clinical and Research Center approved the study. Gemcitabine (GEM)-based, postsurgical adjuvant CTX was allocated to most patients (see online supplementary table S1) by a non-random assignment according to adjuvant protocols in use at the time of surgery. Sixteen patients received a GEM-based neoadjuvant CTX regimen before surgery. The continuous, median and quartile values of the percentages of CD8-TIL and CD68-TAM IRA (immunoreactive area) were compared with demographics, clinical and histopathological features and all these variables were tested as predictors of patient’s outcome (disease-free survival, DFS), patient’s disease-specific survival (DSS) and metachronous metastasis. Further information is listed in the online supplementary files.
Immunohistochemistry and image analysis From each patient enrolled in the study, 2 mm thick tissue slides from formalin-processed and paraffin-embedded tumour sections were retrieved from the archive of the pathology department and processed for immunohistochemistry as previously done.28 Details on immunostaining and image analysis are available in the online supplementary files.
Macrophage cytotoxicity assays To assess macrophage cytotoxicity, coculture experiments of human macrophages and pancreatic cancer cells were performed in the presence or absence of GEM. Details are listed in the online supplementary files.
RESULTS Macrophages accumulate at the tumour–stroma interface in human PDAC and display an immunosuppressive phenotype To evaluate the contribution of TAMs to the immune microenvironment of human PDAC, we systematically and quantitatively assessed by immunohistochemistry the percentage of IRA of CD68+ macrophages (CD68-TAM %IRA) at the tumour–stroma interface (figure 1A and see online supplementary table S1), in 110 tissue specimens from patients with PDAC who underwent surgical resection at our institution. Whole tissue slide quantification evidenced that the density of CD68-TAMs was heterogeneous among patients with PDAC, with some cases showing a high infiltration (figure 1B) and others low infiltration Di Caro G, et al. Gut 2015;0:1–11. doi:10.1136/gutjnl-2015-309193
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Pancreas
Figure 1 CD68-TAMs at the tumour–stroma interface display an immunosuppressive phenotype. (A–D) Histological sections of human PDAC, stained for CD68-TAMs. The image exemplifies the methodology used for the assessment of CD68-TAMs at the tumour–stroma interface (dot line); cancer tissue represents approximately 50% of the microscopic field (A). The density of CD68-TAM cells differs greatly, with some highly infiltrated cases (B) and some scarcely infiltrated cases (C). Distribution of TAM density across 110 patients with PDAC (D). (E–I) TAMs display an immunosuppressive phenotype in human PDAC. Immunohistochemical stainings for CD68 (E), CD163 (F) and CD206 (G) on the same PDAC case and double staining with CD68 and IL-10 (H) antibodies show that TAMs in human PDAC predominantly exhibit an immunoregulatory phenotype. Arrowheads indicate CD68-TAMs expressing IL-10. The majority of macrophages express CD206 (62.4±16.9%, n=12, p=0.003), while IL-12 is expressed only by a minority of macrophages (8.7±10.1%, n=6, p=0.0006). ( J–N) CD68-TAMs in human PDAC localise in regions dominated by an immunosuppressive milieu. TAMs ( J) surrounding a tumour gland expressing the chemokine CCL17 (K), but not CXCL10 (L). CD68-TAMs (M) were also a source of CCL17 (N), associated to a Th2-type response. Arrowheads indicate CD68-TAMs expressing CCL17. Sections in E–G and in J–L and M and N are consecutive sections. Bars: (A) 500 μm, (B and C) 200 μm, ( J–L) 100 μm, (H, M and N) 10 μm. **p=0.003; ***p=0.0006, by Student’s t test. IL, interleukin; %IRA, percentage immunoreactive area; PDAC, pancreatic ductal adenocarcinoma; TAMs, tumour-associated macrophages.
(figure 1C) at the tumour–stroma interface. Figure 1D reports distribution of TAM density across 110 patients with PDAC. Macrophages may have a spectrum of different polarisation states, which dictate their function in the tumour context.10 Accordingly, macrophages at the tumour–stroma interface of pancreatic cancer (figure 1E) predominantly exhibited an immunosuppressive phenotype, as shown by high expression of receptors CD163 (figure 1F), CD206 (figure 1G) and interleukin-10 (IL-10), a prototypical immunosuppressive cytokine (figure 1H). Notably, while expression of CD206 identified most of the CD68+ macrophages present at the tumour–stroma interface (mean%±SD of positive cells 62.4±16.9, n=12, p=0.003), IL-12, which is mainly associated to an M1-like polarisation state in vitro, was expressed only by a minority of macrophages (mean%±SD of positive cells 8.7±10.1, n=6, p=0.0006) (figure 1I), confirming the consolidated evidence that TAMs in human PDAC are prevalently immunosuppressive.11 29 CD68-TAMs (figure 1J) frequently localised in the proximity of Di Caro G, et al. Gut 2015;0:1–11. doi:10.1136/gutjnl-2015-309193
tumour glands expressing the chemokine CCL17 (figure 1K), a Th2-type chemokine, and low or any amounts of CXCL10 (figure 1L), an exemplar interferon (IFN)-γ-induced chemokine, suggesting that CD68-TAMs in human PDAC localise in regions dominated by an immunosuppressive milieu. Notably, CD68-TAMs (figure 1M) were also a source of CCL17 themselves (figure 1N), which is associated to a Th2-type response.
Density of CD68-TAMs at the tumour–stroma interface predicts response to CTX We next analysed the distribution at the tumour–stroma interface and the prognostic value of CD68-TAMs in the microenvironment of human PDAC. In online supplementary table S2, univariate and multivariate Cox regression analyses are shown including demographics, clinical and histopathological features and the density of CD68-TAMs. We recorded 72 (65.7%) outcome events of DFS among 110 patients with PDAC. At multivariate Cox analysis, TNM staging and tumour grade were 3
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Pancreas Table 1 CD68-TAMs as predictors of postsurgical disease-specific recurrences in 110 patients with pancreatic ductal adenocarcinoma, according to CTX treatment Adjuvant CTX treatment No Recurrence rate (%) CD68-TAMs (%IRA)† (quartiles) First 4/12 (33) Second 5/7 (71) Third 4/9 (44) Fourth 7/8 (88)
Yes HR (95% CI)*
p Value
Recurrence rate (%)
HR (95% CI)*
p Value
1.00 2.40 2.29 4.43
0.20 0.26 0.02
13/16 14/20 14/18 11/20
1.00 0.72 0.70 0.37
0.39 0.35 0.01
ref. (0.64 to 8.97) (0.44 to 7.50) (1.21 to 16.24)
(81) (70) (78) (55)
ref. (0.34 to 1.54) (0.33 to 1.50) (0.16 to 0.83)
*Cox regression analysis. †Densities as percentage immunoreactive area at the tumour invasive front. CTX, chemotherapy; TAMs, tumour-associated macrophages.
significantly associated with higher risk of disease recurrence (see online supplementary table S2). Notably, by systematically analysing all the variables that may interact in determining patient prognosis, including the densities of immune cell populations, we found a significant interaction between the density of CD68-TAMs and adjuvant CTX ( p=0.004) (see online supplementary table S2). To assess whether CTX interacted also with the adaptive immune infiltrate, we also evaluated the density of CD8-TILs (CD8-TIL %IRA) (see online supplementary figure S1A and tables S1 and S2). In contrast to CD68-TAMs, densities of CD8-TILs did not show interaction with CTX in predicting the risk of patient’s outcome ( p=0.84) (see online supplementary table S2). Pearson’s linear regression analysis revealed no association between distributions of CD68-TAM %IRA and CD8-TILs %IRA (r=0.07, p=0.49) (see online supplementary figure S1B), suggesting that these immune populations belong to distinct immune networks in the tumour microenvironment. To dissect the interaction of CTX and CD68-TAMs, we designed a subgroup analysis and investigated the prognostic value of CD68-TAMs in patients with PDAC according to the administration of postsurgical CTX (table 1). Surprisingly, while among patients who did not receive any adjuvant CTX (n=36), cumulative increasing values of CD68-TAMs were associated with a higher risk of disease progression (HR=1.57; 95% CI (1.04 to 2.37); p=0.03), among patients with PDAC receiving postsurgical adjuvant CTX (n=74), increasing values of CD68-TAM densities were associated with a better prognosis (HR=0.74; 95% CI (0.58 to 0.95); p=0.02)) (see online supplementary table S1 and figure S2). This result indicates that the prognostic value of TAMs in predicting tumour recurrence is critically influenced by postsurgical
CTX. This evidence was also confirmed by analysing whether response to adjuvant CTX varied according to the density of TAMs at the tumour–stroma interface (table 2). Interestingly, among patients with high densities of CD68-TAMs (fourth quartile, n=28) CTX associated with lower risk of disease recurrence (n=28; HR: 0.38; 95% CI (0.13 to 1.00); p=0.05). Differently, CTX associated with worst prognosis in patients with PDAC with low CD68-TAMs (first to third quartile, n=82) (n=82; HR: 2.19; 95% CI (1.15 to 4.16); p=0.02). This suggests that the presence of CD68-TAMs at the tumour–stroma interface highly influences responsiveness of patients with PDAC to CTX, and could therefore be required for CTX efficacy. Therefore, CD68-TAM assessment could be exploited to identify a subgroup of patients with PDAC highly responsive to CTX and who could receive prognostic advantage from this therapeutic adjuvant treatment. In light of the immunosuppressive phenotype of macrophages we described in PDAC, we tested in our human cohort whether the amount of TAMs at the tumour–stroma interface modulates the prognostic abilities of CD8-TILs after CTX. We found that among patients treated with CTX, the density of CD8-TILs was not a prognostic variable in both patients with low (median) CD68-TAMs (p=0.94, p=0.46) (see online supplementary figure S1C). This result suggests that TAMs are independent of TILs in predicting prognosis and thus their antitumour activities seems to be mediated by T-cell independent pathways. TAMs are known to greatly influence the occurrence of distant metastases.6 30–32 The interaction with postsurgical CTX in patients with PDAC suggests that they might have a role in the suppression of micrometastases, occurring after surgical resection. To investigate this hypothesis, we evaluated the association of TAM density to distant metachronous metastases.
Table 2 Adjuvant CTX treatment as predictor of postsurgical disease-specific recurrences in 110 patients with PDAC, according to the extent of CD68-TAMs at the tumour–stroma interface CD68-TAM (%IRA)* Low (first to third quartile) Recurrence rate (%) Adjuvant chemotherapy treatment No 13/28 (46) Yes 41/54 (76)
High (fourth quartile) HR (95% CI)†
p Value
Recurrence rate (%)
HR (95% CI)†
p Value
2.19 (1.15 to 4.16)
0.02
7/8 (88) 11/20 (55)
0.38 (0.13 to 1.00)
0.05
*Densities as %IRA at the tumour invasive front. †Cox regression analysis. CTX, chemotherapy; %IRA, percentage immunoreactive area; PDAC, pancreatic ductal adenocarcinoma; TAMs, tumour-associated macrophages.
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Pancreas Table 3 CD68-TAMs as predictors of postsurgical distant organ metachronous metastasis in 110 patients with PDAC, according to adjuvant chemotherapy treatment Adjuvant chemotherapy treatment No Metachronous metastasis (%) CD68-TAM (%IRA)† (quartiles) First 3/12 (25) Second 2/7 (29) Third 4/9 (44) Fourth 6/8 (75)
Yes HR (95% CI)*
p Value
Metachronous metastasis (%)
HR (95% CI)*
p Value
1.00 ref. 1.22 (0.20 to 7.34) 2.94 (0.62 to 13.94) 5.10 (1.18 to 22.11)
0.83 0.17 0.02
7/16 10/20 11/18 11/20
1.00 1.01 1.05 0.76
0.97 0.92 0.58
(44) (50) (61) (55)
ref. (0.39 to 2.67) (0.40 to 2.73) (0.29 to 1.99)
*Cox regression analysis. †Densities as percentage immunoreactive area (IRA) at the tumour invasive front. %IRA, percentage immunoreactive area; PDAC, pancreatic ductal adenocarcinoma; TAMs, tumour-associated macrophages.
Significantly, among patients with PDAC who did not receive CTX (n=36), cumulative increasing densities of CD68-TAMs were significantly associated with higher risk of metachronous metastasis (HR=1.77; 95% CI (1.09 to 2.87); p=0.02), while among those who received CTX (n=74) the densities of CD68-TAMs were not associated with prognosis (HR=0.91; 95% CI (0.69 to 1.22); p=0.54) (table 3, see online supplementary figure S2). This result suggests that CD68-TAMs could promote distant metastasis occurrence and that CTX could restrain their prometastatic activity, as evidenced also by Kaplan–Meier prognostic curves (figure 2A, B, see online supplementary figure S2). On the contrary, when we investigated patient’s DSS, we found that TAMs were significantly associated with better survival only among patients who received adjuvant CTX (p=0.05) (figure 2D), while among those untreated, TAM had a tendency to associate to worst prognosis, although not significant (p=0.12) (figure 2C). These results highlight a synergism between CTX and TAMs that differs according to the prognostic clinical endpoint and this could reflect the different biological roles of TAMs in local versus metastatic distant tumour progression.
GEM synergises with TAM-mediated tumour cytotoxicity To provide insight into the interaction between TAMs and CTX, we performed in vitro experiments of tumour cytotoxicity in the presence of differently polarised macrophages and GEM. At an early time point (24 h), treatment of the PDAC cell line (MiaPaCa-2) with 500 mM GEM resulted in a slightly enhanced but not significant tumour cell death (figure 3A, left); however at later time points (48–72 h), the treatment was toxic on tumour cells, confirming that the latter were susceptible to the drug (not shown). M1 macrophages polarised with INF-γ and lipopolysaccharide (LPS) alone did not induce significant increase in PDAC cell cytotoxicity (figure 3A, middle), consistent with the ability of PDAC cells to dampen macrophage antitumour function. By contrast, when GEM was added to the coculture, there was a significant increase in tumour cell death, suggesting that M1 macrophages and CTX have a synergic cytotoxic effect (figure 3A, middle and 3B, left). In contrast, there was a significant decrease in tumour cell death when PDAC tumour cells were cocultured with M2-like macrophages ( polarised with IL-4) compared with PDAC cells alone, suggesting that M2-like macrophages exhibit a protective role towards PDAC tumour cells in vitro ( p≤0.001) (figure 3A, right). However, surprisingly, GEM treatment was able to significantly revert the protective effect of M2-like macrophages (figure 3A, Di Caro G, et al. Gut 2015;0:1–11. doi:10.1136/gutjnl-2015-309193
right and 3B, right panel), reinstating its original cytotoxic effect ( p≤0.01). A dose–response curve with increasing concentrations of GEM showed the specificity of the synergic effect and confirmed that this effect was already induced at low drug concentrations (figure 3C), while at the highest concentration it was not further enhanced. Collectively, these findings reveal that GEM modulates the interaction of macrophages with PDAC cells in vitro and reflect the interaction of GEM and macrophages observed in our human study. We then attempted to dissect the synergism of GEM and macrophages. GEM treatment induced neither selective death of immunosuppressive macrophages (figure 4A) nor tumour cell release of molecules able to activate or reprogram macrophages (figure 4B, C). In contrast, tumour cell death was significantly dose-dependently increased when PDAC tumour cells were cocultured with macrophages pretreated with GEM (MfpreGEM), compared with those cocultured with control macrophages (figure 4B, D), thus suggesting that GEM exerted its modulatory effect by acting directly on the macrophages and thwarting the protective function of macrophages towards PDAC cells. Notably, depending on the cell line tested, the interaction between GEM and macrophages was either abrogated (figure 4D, left panel) or maximum (figure 4D, right panel) at the highest GEM dose. We then investigated the mechanism whereby GEM enhanced macrophage cytotoxicity. Inhibition of macrophage death signals, including tumour necrosis factor (TNF)-α and TRAIL, did not affect the synergism between macrophages and GEM in killing PDAC tumour cells (see online supplementary figure S3). In contrast, GEM treatment induced an early and dramatic increase in cellular reactive oxygen species (ROS) in macrophages (figure 4E), thus suggesting that release of cytotoxic ROS could be responsible for the induced macrophage antitumour activity.
GEM directly modifies macrophage polarisation in vitro and in the tumour microenvironment of human PDAC To reveal molecular actors regulating the CTX-mediated modulation of macrophage function, we evaluated the effect of GEM on the mRNA expression profile of macrophages. GEM-treated macrophages significantly increased expression of several immune-stimulating cytokines, such as IL-12, IL-27, IFNG, as well as cytokines typically involved in inflammatory responses, including prostaglandin-endoperoxide synthase, Fas ligand and TNF-α (figure 5A). In accordance with the evidence that GEM induces ROS production in macrophages (figure 4E), there was also an increase in molecules regulating microbial responses such as inducible nitric oxide synthase and nicotinamide 5
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Pancreas
Figure 2 Clinical relevance of CD68-TAMs in human pancreatic adenocarcinoma. (A and B) Kaplan–Meier curves showing metachronous metastasis, according to CD68-TAM density and CTX. A high density (≥median) of CD68-TAMs associated to an increased risk of distant tumour metastasis compared with a lower density only in patients who did not receive adjuvant CTX ( p=0.02, n=36) (A), but not in those CTX treated ( p=0.68, n=74) (B). (C and D) Kaplan–Meier curves showing DSS%, according to CD68-TAM density and CTX. A high density (≥median) of CD68-TAMs was significantly associated with better survival only among patients who received adjuvant CTX ( p=0.05) (C), while there was a tendency to associate to worst prognosis, but not significant in untreated patients ( p=0.12) (D). CTX, chemotherapy; DSS, disease-specific survival; TAMs, tumour-associated macrophages.
adenine dinucleotide phosphate, although not significant. Remarkably, the increase in proinflammatory mediators was paralleled by a significant decrease in IL-10, also confirmed by decreased protein production (see online supplementary figure S5), suggesting an overall switch in activation status. In accordance with these results, the gene expression profile of GEM-treated macrophages also evidenced a significantly decreased expression of some growth factors, such as platelet growth factor A and B, placental growth factor and matrix proteins, such as osteopontin and fibronectin (figure 5A), which are mediators typically upregulated in protumour M2-like TAM. Overall, these findings strongly suggest that GEM treatment acts directly on macrophages by inducing a downregulation of trophic factors, concurrently inducing an increased expression of inflammatory mediators able to revert this protective function and likely to explain the protumour activity of CTX-untreated macrophages. In accordance with gene expression data, GEM treatment significantly modified surface expression of polarising markers, upregulating expression of the M1-markers HLA-DR, CD40 and the chemokine receptor CCR7 and downregulating the M2-markers CD163 and CD206 (figure 5B, C). To further confirm the effect of CTX on macrophage repolarisation, we took advantage of a cohort of consecutive patients who were administered neoadjuvant CTX and evaluated the phenotype of macrophages at the tumour front, by immunohistochemistry on tumour tissue slides. The density of CD68-TAMs and CD8-TILs at the tumour–stroma interface was not significantly modified after neoadjuvant CTX, although a slight tendency to decreased density of CD68-TAMs was 6
observed ( p=0.11, see online supplementary table S1). However, despite a comparable density, the phenotype of macrophages at the tumour–stroma interface was affected by CTX administration: while in untreated patients a considerable amount of CD68-TAMs expressed the M2-marker CD206 (figure 5D, upper panel), significantly less macrophages were CD206-positive in PDAC sections from neoadjuvant-treated patients (figure 5D, lower panel and 5E). Similarly, while the majority of macrophages in human PDAC was IL-10+, in patients treated with CTX the presence of IL-10+ macrophages decreased significantly ( p
