Chemotherapy-induced infiltration of neutrophils promotes pancreatic cancer metastasis via Gas6/AXL signalling axis

Background

Metastasis is the leading cause of cancer-related death. Pancreatic ductal adenocarcinoma (PDAC) frequently metastasizes to the liver1 2 and liver metastasis is accompanied by the formation of an inflammatory-fibrotic metastatic microenvironment that supports the colonisation and outgrowth of disseminated cancer cells.3–6 Myeloid immune cells, including monocytes, macrophages and neutrophils, are found in high numbers in the metastatic niche and have been shown to promote the metastatic process.7–9 Macrophages are highly plastic cells and, depending on their activation state, can acquire tumour supportive or tumour repressive functions.10 11 During liver metastasis, macrophages are prometastatic, display an immunosuppressive phenotype,12 13 and promote fibrosis.3 Emerging evidence suggests that neutrophils play a critical role during the early steps of metastasis.14 Neutrophils can promote the colonisation of the distant site through the release of neutrophil extracellular traps (NETs),15 16 induction of angiogenesis,9 17 secretion of leukotrienes18 and by their immunosuppressive activities.19 20 However, whether myeloid immune cell functions in pancreatic cancer metastases are altered in response to therapeutic interventions remains unknown.

Systemic spread is an early event in pancreatic cancer progression1 and by the time PDAC patients are diagnosed, the majority (~80%) present with non-resectable metastatic cancer.2 A total of 15%–20% of PDAC patients are eligible for surgical resection of their primary tumour. However, clinically undetectable micrometastatic lesions are often already present at the time the primary tumour is removed, and more than 70% relapse with distant metastasis within 24 months of surgery.21 The time of recurrence after surgical resection strongly correlates with overall survival, and an early hepatic metastatic relapse is associated with the worse prognosis.22 Following diagnosis of liver metastases, median survival on systemic chemotherapy is just 9 months.23

Cytotoxic chemotherapy is the standard care of treatment for all patients with pancreatic cancer, including those with locally advanced or metastatic disease and as adjuvant treatment for patients after surgical resection of their primary tumour.24 Gemcitabine, gemcitabine/capecitabine, nab-paclitaxel and FOLFIRINOX are the most common chemotherapeutic treatment options.25 Although the effect of chemotherapy on the primary tumour site is well characterised,21 our understanding of how chemotherapy shapes the hepatic metastatic microenvironment and how this affects metastatic disease progression remains unknown. A better understanding of this process could lead to treatments that improve the efficacy of current systemic chemotherapies.

ResultsGemcitabine treatment restrains metastatic progression, but disease relapses when treatment is withdrawn

To model chemotherapeutic treatments of metastatic pancreatic cancer in vivo, we induced PDAC liver metastasis in mice by intrasplenic implantation of KPC derived cells and initiated gemcitabine treatment once metastatic lesions had been established (at day 12 postimplantation),3 (figure 1A). While KPC cancer cells were sensitive to gemcitabine when treated in vitro (online supplemental figure S1A), gemcitabine treatment did not improve the overall survival of animals with pancreatic cancer liver metastasis (figure 1A). Bioluminescent in vivo imaging analysis revealed that metastatic tumour burden was significantly reduced in the gemcitabine treated animals at the end of the treatment schedule (d22) (figure 1B), but no differences in tumour burden were detected at the humane endpoints (between day 32 and day 48 (figure 1C). H&E staining of liver tissue sections further confirmed a significant reduction of metastatic tumour lesions by the end of the treatment schedule (day 22), while this reduction was no longer detected at humane endpoints (online supplemental figure S1B–E). Postmortem analysis proofed extensive tumour burden in the liver, while tumour formation in the spleen remained minor (online supplement figure S1F, G). We next assessed tumour cell death in livers from control (saline treated) versus gemcitabine treated animals. We found that the percentage of apoptotic cancer cells, assessed by cleaved caspase 3 (CC3) staining, was significantly increased in gemcitabine treated animals compared with control tumour-bearing mice by the end of the treatment schedule (day 22) (figure 1D,E). However, by humane endpoints, after withdrawal of gemcitabine treatment, the initially observed increase in cancer cell death was lost (online supplemental figure S1H, I). Adjuvant chemotherapy is the established standard-of-care for patients who undergo surgical resection of their primary pancreatic tumour.24 The short time and the high frequency at which these patients relapse with metastatic disease (median of 9 months after resection23) strongly suggests that occult micrometastases were already established at the time of surgery.26 To test the effect of chemotherapy on micrometastatic lesions equivalent to the adjuvant treatment setting, we next administered a single dose of gemcitabine at day 3 post-tumour implantation, after the initial seeding period and where micro-metastatic lesions are present (figure 1F).3 13 Similar to what we observed with larger metastatic lesions, administration of gemcitabine also reduced micro-metastatic tumour burden (figure 1G,H) and tumour lesion areas in the liver at day 4 (figure 1I,J), but this effect was lost at day 14 (figure 1G–J). By 24 hours after gemcitabine administration, the percentage of apoptotic cancer cells (TUNEL+) in micrometastatic lesions was markedly increased in gemcitabine treated animals compared with control animals (figure 1K,L). However, at day 14, the rate of TUNEL +cancer cells declined in the gemcitabine treated tumours to similar levels as in the untreated cohort (online supplemental figure S1J, K). Macrophages and dendritic cells (DCs) are phagocytic cells from the innate arm of the immune system that play a key role in the removal of dead cells and are critical for the induction of an antitumour immune response in cancer.27 In order to identify the phagocytosis of cellular cancer debris by macrophages and DCs, we used flow cytometry to measure the fluorescent signal of zsGreen labelled cancer cells within macrophages (CD11b+F4/80+CD11cneg), CD11b+ DCs (CD11c+CD11b+CD103negF4/80neg) and CD103+ DCs (CD11c+CD103+CD11bnegF4/80neg). All three cell populations showed a significant uptake of zsGreen signal after gemcitabine administration compared with the control group (figure 1M; online supplemental figure S1L), suggesting an activation of innate immune cells in metastatic lesions in response to chemotherapy. Taken together, these findings show that gemcitabine induces cancer cell death in PDAC metastatic lesions, but tumour growth relapses after treatment withdrawal and overall survival remains unchanged.

Figure 1Figure 1Figure 1

Gemcitabine restrains metastatic progression during treatment, but disease relapses and overall survival remain unchanged when treatment is withdrawn. (A–E) Liver metastasis was induced by intrasplenic implantation of 1×106 KPCluc/zsGreen cells. Starting day 12, animals were treated with gemcitabine (100 mg/kg) or control (vehicle) every 3 days with four doses in total. (A) Survival analysis of gemcitabine and control-treated mice-bearing liver metastasis; log-rank (Mantel-Cox) test, p=0.2499. Median survival for control was 22 days (n=6 mice) and gemcitabine 33.5 days (n=8 mice) after treatment initiation. (B) Representative images of bioluminescence imaging (BLI) taken 1 day after last treatment dose (day 22). (C) Tumour burden assessed by BLI in gemcitabine treated group (n=8 mice) compared with control group (n=6 mice) at day 22 and humane endpoint (HEP). (D, E) Representative immunofluorescent images (D) and quantification (E) of apoptotic KPCluc/zsGreen cells staining positive for cleaved caspase 3 (CC3) at day 22 (n=5 mice /group). White arrowheads indicate apoptotic (CC3+) cancer cells. (F–L) Liver metastasis was induced by intrasplenic implantation of 5×105 KPCluc/zsGreen cells and animals received one dose of gemcitabine (100 mg/kg) or control (vehicle) at day 3 (F). (G, H) Representative BLI images of dissected livers (G) and change in tumour burden (H) (day 4: n=5 mice/group/time point). (I, J)Representative images of H&E-stained liver sections (I) and quantification (J). (K, L)Rrepresentative immunofluorescent images of apoptotic KPCluc/zsGreen cells staining positive for TUNEL at day 4 (n=5 mice/group) (K) and quantification (L).White arrowheads indicate apoptotic (TUNEL+) cancer cells. (M) uptake of apoptotic zsGreen-labelled KPC FC1199luc/zsGreen cancer cells by dendritic cells (DC) and macrophages (MACS) was evaluated 1 day after gemcitabine treatment. Frequency of zsGreen +cells among CD103+ DC, CD11b+ DC and MACS (n=5 mice/group). Scale bar 50 µM. Data are presented as mean±SEM. Unpaired t-test was used to calculate p values. *P<0.05; **p<0.01. H, healthy liver; M, metastases; n.s., not significant.

Gemcitabine treatment induces a short-term activation of a proinflammatory immune response in metastatic hepatic lesions

Since chemotherapy can promote the activation of an immune response in cancer27 we next investigated, in more detail, the immune cell activation on gene expression level in metastatic lesions during the initial response to gemcitabine treatment (day 4) and after withdrawal (day 14) using the Mouse PanCancer Immune Profiling Panel (NanoString Technologies). Hierarchical clustering of the generated pathway scores revealed that gemcitabine induces distinct transcriptional changes during the initial response, as highlighted by the separate clustering of the gemcitabine groups compared with control groups (figure 2A, left). However, the distinct signatures between control and gemcitabine-treated metastatic lesions were lost after withdrawal, as indicated by the loss of segregation between the two groups (figure 2A, right). Among the top upregulated pathways, we identified innate immune activation and T cell functions which are characteristic of an antitumour immune response (figure 2B). However, after gemcitabine withdrawal, these immune stimulatory pathways were markedly downregulated, suggesting that gemcitabine only triggers a temporal activation of an anti-tumour immune response in tumour-bearing mice (figure 2C). We next analysed disaggregated metastatic lesions by mass and flow cytometry to assess immune cell infiltration and their activation state. We found that during the initial response, macrophage numbers (CD45+CD11b+Ly6GnegF4/80+) significantly increased, and inflammatory monocytes numbers (CD45+CD11b+Ly6ChighLy6GnegF4/80neg) were reduced (figure 2D; online supplemental figure S2A, B). In addition, CD4+ T cell numbers significantly increased in response to treatment (online supplemental figure S3A, C). However, after chemotherapy withdrawal, neutrophils (CD45+CD11b+F4/80negLy6G+) and patrolling monocytes (pMo; CD45+CD11b+Ly6ClowF4/80low/negMHCIIneg) increased the most in gemcitabine-treated tumours compared with control treated tumours, while T cell numbers were significantly decreased (figure 2E). The decrease in total T cell numbers was most likely due to a reduction in CD8+ T cells, since the less abundant CD4+FoxP3+ T regulatory cells (Tregs) rather increased (online supplemental figure S3A–D). Consistent with an antitumour immune response, we found a significant increase in the activation of CD8+ and CD4+ T cells (figure 2F; online supplemental figure S3E, F), DCs (figure 2G), macrophages (figure 2H) and NK cells (figure 2I) during the initial response to gemcitabine (online supplemental figure S2A, B). Again, this effect was lost after withdrawal of the treatment.

Figure 2Figure 2Figure 2

Gemcitabine administration induces a short-term activation of a proinflammatory immune response in metastatic pancreatic cancer. (A–K) Liver metastasis was induced by intrasplenic implantation of KPCluc/zsGreen cells and animals were treated with gemcitabine (100 mg/kg) or control (vehicle) at day 3. Metastatic livers were resected at initial response (day 4) and after withdrawal of treatment (day 14) for transcriptional, mass cytometry and tissue staining analysis. (A) Heatmap depicting hierarchical clustering of pathway scores (n=3 mice/group/time point). (B–C) Graph depicting top pathway scores observed in (B) metastatic livers of gemcitabine treated animals compared with control animals during initial response (day 4) and in (C) metastatic livers after gemcitabine withdrawal (day 14) compared with the initial response (day 4). (D, E) Coloured viSNE maps with each colour representing one immune cell population assessed by mass cytometry and quantification of main immune cell types among control (CTR) and gemcitabine (GEM) treated liver metastases at day 4 (A) and day 14 (B), respectively (CTR D4 n=4 mice, GEM D4 n=4 mice; CTR d14 n=3 mice; GEM d14 n=4 mice). (F–I) Quantification of metastasis infiltrating immune cells and their activation state by mass cytometry at initial treatment response (day 4) and after treatment withdrawal (day 14). (F)Cytotoxic CD8+ T cell activation (CD69+), (G) dendritic cell (DC) activation (CD86 +MHCIIhigh), (H) macrophage activation (CD86 +MHCIIhigh) and (I) natural killer (NK) cell activation (CD69+) (CTR D4 n=4 mice, GEM D4 n=4 mice; CTR d14 n=3 mice; GEM d14 n=4 mice). (J, K) Representative immunofluorescent images and quantification of iNOS+ and F4/80+ macrophages in liver tumours during initial response (n=4 mice/group) (D) and after gemcitabine withdrawal (E) (n=3 mice in CTR group; n=4 mice in GEM group). White arrowheads indicate iNOS+ macrophages. Scale bar 50 µM; M=metastases, H=healthy liver. Data are presented as mean±SEM. *P<0.05; **p<0.01; n.s., not significant, by unpaired t-test. For multiple comparisons (D, E), one-way ANOVA coupled with Dunnett’s post hoc testing was performed. ANOVA; analysis of variance.

Further analysis of metastatic liver tissues confirmed that gemcitabine treatment induces the overall accumulation of macrophages (F4/80+), particularly of macrophages with a proinflammatory phenotype (iNOS+) (figure 2J) while macrophages with an immunosuppressive phenotype (CD206+ and YM1+) were reduced (online supplemental figure S3G-J). However, no significant changes in macrophage infiltration or activation were observed after treatment withdrawal (figure 2K; online supplemental figure S3K, L). Taken together these data suggest that gemcitabine administration induces the activation of an antitumourigenic immune response at the metastatic site, characterised by an increase in proinflammatory macrophages, activated CD8+ T cells and NK cells. However, after treatment withdrawal the initial immune cell activation is lost and metastatic lesions revert back to an immunosuppressive microenvironment, which is commonly found in established metastatic PDAC tumours.12 13

Macrophage depletion after gemcitabine treatment increases CD8+ T cell infiltration, but neutrophil depletion has no effect on CD8+ T cell numbers

Since neutrophils and macrophages can both effectively suppress CD8+ T cell responses,11 14 we next questioned whether the depletion of either of these myeloid cell types is sufficient to stop metastatic relapse and to sustain the initially observed CD8+ T cell response (figure 2B and F). To address this question, we ran two separate depletion studies using monoclonal antibodies targeting neutrophils (αLy6G) or macrophages (αCSF-1) in the presence or absence of gemcitabine treatment. Liver metastasis was induced by intrasplenic implantation of KPC cells. After 3 days, mice-bearing micrometastatic lesions were treated with gemcitabine or saline (control) and 1 day later (day 4) we commenced the depletion of neutrophils (figure 3A–C) or macrophages (figure 3D–F) using αLy6G and αCSF-1 antibodies, or their corresponding isotype controls (IgG). Depletion of neutrophils after gemcitabine administration markedly reduced the metastatic tumour burden compared with gemcitabine/IgG treatment (figure 3B,C, online supplemental figure S4A, B), but depletion of neutrophils in the absence of gemcitabine did not have any effect on metastatic tumour burden (online supplemental figure S4C). In contrast, depletion of macrophages by αCSF-1 significantly reduced metastatic tumour burden in both saline (control) and gemcitabine treated mice (figure 3E,F; online supplemental figure S4D, E). Flow cytometry analysis of disaggregated metastatic lesions derived from gemcitabine treated animals revealed that the depletion of macrophages increased CD8+ T cell infiltration, while neutrophil depletion did not affect CD8+ T cell infiltration (figure 3G). In agreement with these findings, we did not detect an increase in Granzyme B expression in CD8+ T cells in gemcitabine-treated animals where neutrophils were depleted (figure 3H), but we found a significant increase of Granzyme B expressing CD8+ T cells in metastatic lesions of gemcitabine-treated mice where macrophages were depleted (figure 3J,K). We also confirmed that applied macrophage-depletion and neutrophil-depletion strategies indeed reduced their corresponding numbers at the metastatic site (online supplemental figure S4F–H). Notably, neutrophil-depletion or macrophage-depletion after gemcitabine treatment also increased overall survival of the mice compared with gemcitabine treatment alone (figure 3L,M). Taken together, these data show that gemcitabine administration is accompanied by an infiltration of macrophages during the initial response, while neutrophils are recruited to the metastatic site after therapy withdrawal. Depletion of macrophages or neutrophils after gemcitabine withdrawal enhances the therapeutic effect of gemcitabine. Notably, while macrophage depletion restores CD8+ T cell infiltration and activation, neutrophil depletion does not affect CD8+ T cells, suggesting that neutrophils promote metastatic relapse in a CD8+ T cell independent manner.

Figure 3Figure 3Figure 3

Macrophage depletion increases CD8+ T cell infiltration, but neutrophil depletion has no effect on CD8+ T cell numbers. (A–M) Liver metastasis was induced by intrasplenic implantation of KPCluc/zsGreen cells and animals were treated with gemcitabine (100 mg/kg) or control (vehicle) at day 3. (A–C, G–I) At day 4, mice were treated with IgG control (CTR) or αLy6G antibody. Schematic illustrating experiment (A). Change in tumour burden was quantified by in vivo BLI (n=3 mice/group). Representative images (B) and quantification (C). (D–G, J, K) At day 4, mice were treated with IgG control (CTR) or αCSF-1 antibody. Schematic illustrating experiment (D). Change in tumour burden was quantified by in vivo BLI (CTR n=3 mice; αCSF-1 n=4 mice). Representative images (E) and quantification (F). (G) Change in CD8+ T cell infiltration into metastatic lesions was quantified by flow cytometry analysis in mice treated with αLy6G or αCSF-1 or their corresponding IgG controls. (H, I) Representative immunofluorescent images of CD8+GranzymeB+ T cell staining of liver sections from mice treated with IgG or αLy6G (H) and quantification (I) of CD8+GranzymeB+ T cells (GranzymeB=GzmB). (J, K) Representative immunofluorescent images of CD8+GranzymeB+ T cell staining of liver sections from mice treated with IgG or αCSF-1 (J) and quantification (K) of CD8+GranzymeB+ T cells. White arrowheads indicate CD8+GranzymeB+ T cells. (L) Liver metastasis was induced by intrasplenic implantation of 1×106 KPCluc/zsGreen cells. At day 3, all animals were treated with gemcitabine (100 mg/kg). At day 4, mice were treated with IgG control (CTR) or αLy6G antibody. survival analysis of gemcitabine + IgG and gemcitabine + αLy6G antibody-treated mice-bearing liver metastasis; log-rank (Mantel-Cox) test, p=0.0022. Median survival for gemcitabine + IgG was 35 days (n=6 mice) and gemcitabine + αLy6G 48 days (n=6 mice). (M) same as (L), but at day 4, mice were treated with IgG control (CTR) or αCSF-1R antibody. Survival analysis of gemcitabine + IgG and gemcitabine + αCSF-1R antibody-treated mice-bearing liver metastasis; log-rank (Mantel-Cox) test, p=0.0168. Median survival for gemcitabine + IgG was 29.5 days (n=6 mice) and gemcitabine + αCSF-1R 45 days (n=6 mice). Scale bar 50 µM. Data are presented as mean±SEM. Unpaired t-test was used to calculate p values. *P<0.05; **p<0.01. H, healthy liver; M, metastases; n.s., not significant.

Chemotherapy withdrawal triggers the recruitment of Gas6-expressing neutrophils to hepatic metastatic tumours

Next, we further explored the mechanism by which neutrophils promote metastatic relapse after chemotherapy withdrawal. Neutrophil depletion after gemcitabine treatment reduced metastases in the presence and absence of CD8+ T cells (figure 4A; online supplemental figure S4I, J), suggesting that the neutrophils can directly affect cancer cell regrowth. Hence, we next assessed cancer cell proliferation in tumour sections after gemcitabine withdrawal (day 14). After gemcitabine withdrawal, metastatic deposits showed a significant increase of proliferating (Ki67+) cancer cells compared with metastatic deposits from the saline treated control group (figure 4B,C). Importantly, the depletion of neutrophils only reduced cancer cell proliferation associated with gemcitabine withdrawal, and had no impact on cancer cell proliferation in saline (control) treated mice, suggesting a treatment induced growth promoting function of neutrophils (figure 4B,C). Thus, to test this hypothesis, we isolated metastasis infiltrating neutrophils from metastatic livers from mice treated with saline (control) or gemcitabine, and cocultured them with gemcitabine pretreated pancreatic cancer cells under anchorage independent growth conditions ex vivo. Gemcitabine-treated pancreatic cancer cells were unable to form colonies (figure 4D; online supplemental figure S4K). Strikingly, coculturing of neutrophils isolated from gemcitabine treated metastatic livers with gemcitabine-treated cancer cells enabled the cancer cells to grow and form colonies, while neutrophils isolated from control (saline treated) metastatic lesions were unable to promote cancer cell proliferation (figure 4D). In contrast, while metastases derived macrophages were also able to significantly increase cancer cell colony formation, the macrophage-growth promoting functions were Gas6-independent, unaffected by gemcitabine and markedly less potent compared with neutrophils derived from gemcitabine treated mice (figure 4D; online supplemental figure S4L). In agreement with these findings, in vivo, Ki67+ cancer cell numbers were reduced in macrophage depleted mice independent of their treatment (online supplemental figure S4M, N). Taken together, these data show that gemcitabine treatment makes neutrophils acquire a promitogenic capacity that promotes cancer cell proliferation.

Figure 4Figure 4Figure 4

Neutrophils promote cancer cells proliferation and Gas6 is highly expressed by metastatic associated neutrophils after gemcitabine treatment. (A) Liver metastasis was induced by intrasplenic implantation of KPCluc/zsGreen cells and animals were treated with gemcitabine (GEM; 100 mg/kg) at day 3. At day 4, mice were treated with αLy6G or IgG controls for 2 weeks; at day 7, mice were treated with αCD8 or IgG controls until end point (day 14). Change in tumour burden was quantified by ex vivo bioluminescence imaging (BLI) (n=5 mice/group). (B–C) Liver metastasis was induced by intrasplenic implantation of KPCluc/zsGreen cells. Mice were treated with gemcitabine or saline 3 days postcell implantation. Treatment with αLy6G or control IgG started at D4 (n=4 mice/group). Livers were resected after 14 days and assessed by Ki67 staining (proliferation marker). Representative IHC images (B) and quantification of proliferating Ki67+ tumour cell frequency in metastatic livers (C). Inset: asterisks indicate ductal structures formed by metastatic tumour cells (red arrow head). (D)Colony formation assay of gemcitabine stressed KPC cells in the presence or absence of metastasis infiltrating neutrophils (+Ly6G) or macrophages (+F4/80) isolated from tumour-bearing livers of mice at day 14 after treatment with GEM or saline treated (CTR). Bar graph shows fold upregulation of BLI signal compared with Gem-treated KPC cells alone (red shaded) (three independent experiments; mean±SEM). (E) Quantification of Gas6 mRNA levels by real time PCR in intrametastatic pancreatic cancer cells, neutrophils (Ly6G), macrophages (F4/80) and non-immune stromal cells (zsGreennegCD45neg), isolated by fluorescence activated cell sorting from established metastatic livers at day 14 after treatment with GEM or untreated (CTR). Bar graph shows relative expression of Gas6 in cells derived from GEM-treated mice and untreated mice (data are from three independent experiments; mean±SEM). (F–H) Representative images (F) of myeloperoxidase (MPO) and Gas6 staining using RNAscope in serial sections from metastatic livers derived from untreated (CTR) or GEM treated mice (n=3 mice/group). Arrowheads indicate Gas6+ staining in neutrophil-rich areas. Scoring of Gas6 signal per field of view (G) and MPO staining quantification (H). (I–K) Metastatic tumours in livers of the spontaneous mouse pancreatic cancer model KrasG12D;Trp53R172H;Pdx1-Cre (KPC mice) treated with Gemcitabine (KPC Gem) or left untreated (KPC Ctr) were isolated and analysed (n=3 mice/group). Representative images (I) of MPO and Gas6 staining using RNAscope in serial sections from metastatic tissue sections. Arrowheads indicate Gas6+ staining in neutrophil-rich areas. (J) Scoring of Gas6 signal per field of view and (K) MPO staining quantification. (L, M) Peripheral blood neutrophils were isolated from metastatic PDAC patients during their first cycle of gemcitabine treatment and GAS6 mRNA levels were assessed by real time PCR. Schematic illustration of treatment regimen and patient blood sample collection (L). Quantification of data (M) (BL=baseline, prior treatment) (n=2 patients). Scale bar=50 µM. Data are presented as mean±SEM. Unpaired t-test or ANOVA with Bonferroni was used to calculate p values. *P<0.05; **p<0.01; ***p<0.001. ANOVA; analysis of variance; H, healthy liver; IHC, immunohistochemistry; M, metastases; n.s., not significant; PDAC, pancreatic ductal adenocarcinoma.

We next aimed to understand how neutrophils promote cancer cell proliferation. To achieve this goal, we performed RNA sequencing of metastasis infiltrating neutrophils isolated from saline treated metastatic livers (Ly6GCtr) and gemcitabine treated metastatic livers (Ly6GGem) (online supplemental figure S4O). Differently expressed genes were first filtered for GO terms extracellular and receptor ligand activity. Among the resulting n=141 genes, we identified Growth Arrest Specific protein 6 (Gas6) as one of the top upregulated genes in neutrophils derived from gemcitabine treated metastatic lesions compared with control metastatic lesions (online supplemental tables S1, S2). Gas6 and its main receptor AXL are overexpressed in pancreatic cancer and their expression correlates with poor prognosis.28 29 Gas6/AXL signalling in cancer cells is associated with tumour cell proliferation, epithelial mesenchymal transition and metastases.30 31 Subsequent analysis of Gas6 expression in flow cytometry sorted neutrophils, non-immune stroma cells, macrophages and cancer cells confirmed that within the metastatic tumour microenvironment, neutrophils markedly upregulate Gas6 expression in response to gemcitabine treatment and neutrophils are the main source of Gas6 after gemcitabine withdrawal (figure 4E; online supplemental figure S5A).

In agreement with these findings, we found a marked upregulation of Gas6 expression levels in neutrophil-rich areas proximate to cancer cells after gemcitabine withdrawal in serial tissue sections derived from experimental (figure 4F–H; online supplemental figure S5B, D) and spontaneous hepatic metastatic lesions (figure 4I–K; online supplemental figure S5C, D). In contrast, chemotherapy withdrawal did not increase Gas6 levels in metastatic livers from neutrophil-depleted mice (online supplemental figure S5E, F). Moreover, we only found an increase of Gas6 expressing neutrophils in tumour-bearing livers, but not in tumour-free lung tissues, suggesting Gas6 expressing neutrophils preferentially accumulate at the metastatic tumour site (online supplemental figure S5G, H).

We observed the same changes when we treated metastatic tumour-bearing mice with nab-paclitaxel or FOLFIRINOX, both commonly used chemotherapy regimens in PDAC25 (online supplemental figure S5I). With all chemotherapeutic treatments, metastatic tumour burden temporarily decreased but was followed by metastatic relapse (online supplemental figure S5J) which was accompanied by an influx of Gas6-expressing neutrophils into metastatic lesions (online supplemental figure S5K–N). These results suggest that the increased accumulation of Gas6-expressing neutrophils in relapsed metastatic lesions after chemotherapy treatment occurs in response to different chemotherapeutic treatment regimens and is therefore not agent specific. We next assessed Gas6 expression in circulating neutrophils in patients with metastatic pancreatic cancer and in our mouse metastases model. We collected patient blood samples prior (baseline) and after (week 4) their first cycle of gemcitabine treatment (figure 4L). We found that Gas6 expression increased in circulating neutrophils 4 weeks after the first dose of treatment (figure 4M; online supplemental figure S6A). Similarly, in the preclinical mouse model, Gas6 expression was increased in circulating murine neutrophils after gemcitabine withdrawal (online supplemental figure S6B). Since the release of NETs by apoptotic neutrophils has been shown to promote pulmonary metastatic outgrowth in breast cancer models,16 we also analysed the presence of apoptotic (TUNEL+) neutrophils in liver metastases. However, we could only detect a few apoptotic neutrophils within liver metastases and their numbers remained unaffected by gemcitabine treatment (online supplemental figure S6C, D). To assess the biological importance of Gas6 in promoting regrowth of metastatic cancer cells, we next isolated metastasis infiltrating neutrophils from gemcitabine-treated tumour-bearing mice (Ly6GGem) and cocultured those neutrophils with gemcitabine treated cancer cells in

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