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British Journal of Cancer logoLink to British Journal of Cancer
. 2022 Jun 20;127(6):1142–1152. doi: 10.1038/s41416-022-01886-4

M-CSF as a therapeutic target in BRAFV600E melanoma resistant to BRAF inhibitors

C Barceló 1,#, P Sisó 1,#, I de la Rosa 1, C Megino-Luque 1,2, R Navaridas 1, O Maiques 3, I Urdanibia 1, N Eritja 1,2, X Soria 4, M Potrony 5,6, N Calbet-Llopart 5,7, S Puig 5,7, X Matías-Guiu 2,8,9, R M Martí 2,4,✉, A Macià 1,10,✉
PMCID: PMC9470708  PMID: 35725813

Abstract

Background

Disseminated BRAFV600E melanoma responds to BRAF inhibitors (BRAFi) but easily develops resistance with poor prognosis. Secretome plays a pivotal role during tumour progression causing profound effects on therapeutic efficacy. Secreted M-CSF is involved in both cytotoxicity suppression and tumour progression in melanoma. We aimed to analyse the M-CSF contribution in resistant metastatic melanoma to BRAF-targeted therapies.

Methods

Conditioned media from melanoma cells were analysed by citoarray. Viability and migration/invasion assays were performed with paired melanoma cells and tumour growth in xenografted SCID mice. We evaluated the impact of M-CSF plasma levels with clinical prognosis from 35 metastatic BRAFV600E-mutant melanoma patients.

Results

BRAFi-resistant melanoma cells secretome is rich in pro-tumour cytokines. M-CSF secretion is essential to induce a Vemurafenib-resistant phenotype in melanoma cells. Further, we demonstrated that M-CSF mAb in combination with Vemurafenib and autophagy blockers synergistically induce apoptosis, impair migration and reduce tumour growth in BRAFi-resistant melanoma cells. Interestingly, lower M-CSF plasma levels are associated with better prognosis in metastatic melanoma patients.

Conclusions

Secreted M-CSF induces a BRAFi-resistant phenotype and means worse prognosis in BRAFV600E metastatic melanoma patients. These results identify secreted M-CSF as a promising therapeutic target toward BRAFi-resistant melanomas.

Subject terms: Melanoma, Prognostic markers

Introduction

Melanoma is one of the most aggressive and deadly type of skin cancer [1]. Advanced malignant melanoma has a poor prognosis and treatment does not always lead to an effective response [2]. BRAF mutation are the most frequent in melanoma (40–50% cases) and leads to the activation of an MEK-ERK pathway that induces proliferation and tumour progression of melanoma cells [3]. Notably, targeting BRAFV600E melanomas with BRAF inhibitors (BRAFi), such as Vemurafenib (Vem), in combination with MEK inhibitors (MEKi), reduces cell proliferation and melanoma progression. Their major limitation, however, is the rapid development of resistance mechanisms [4]. Despite great advances in the targeted-therapy field and immunotherapy, the treatment of melanoma is still a clinical challenge.

Macroautophagy (hereinafter autophagy) is a highly conserved and constitutively induced self-degradative process in melanoma cells [5, 6], which contributes to melanoma initiation, progression, and targeted-therapy resistance [6–11]. Previous studies have shown a positive correlation between Cav3.1 T-type calcium channel (TTCC) isoform and LC3II expression, which is enhanced in BRAFV600E-mutant primary and metastatic melanomas. Furthermore, Cav3.1 levels, coupled with autophagy, increase gradually during Vemurafenib-resistance acquisition in melanoma cells, which act as intrinsic mechanisms of drug resistance [6, 9, 10]. TTCC blockers inhibit autophagy and induce cell death in all melanoma cells [5, 6]. Specifically, in BRAFV600E-mutant melanoma cells, TTCC blockers as well as Chloroquine (CQ) inhibit migration and invasion processes due to autophagic blockade [6]. TTCC blockers targeting Cav3.1 isoform could be an alternative therapeutic strategy for BRAFi-resistant melanomas [6, 9, 10].

Tumour microenvironment (TME) plays a critical role in the progression of malignant neoplasms, from early phases to advanced disease [12]. The TME exerts an antitumour effect at early stages [13], but melanoma cells progressively acquire several immune escape mechanisms, including the ability to modulate TME to suppress the antitumour immune cell response [14]. Such suppression is partially induced by extracellular proteins secreted by melanoma cells (secretome) [15, 16]. Secretome acquires the ability to suppress the antitumour immune cell response, favours melanoma progression and metastasis and even has a profound effect on therapeutic efficacy [13, 17]. BRAFi-treated melanoma cells have been shown to develop a complex and reactive secretome (therapy-induced secretome), which stimulates the expansion and spread of drug-resistant clones [18–20]. Moreover, the continuous crosstalk between tumour cells and their surrounding stroma results in environment-mediated drug resistance [12, 21].

M-CSF (macrophage colony-stimulating factor) is a cytokine secreted by many cell types, such as tumour cells, including melanoma [22–25]. M-CSF is involved in both cytotoxicity suppression and promoting pro-tumour effects, such as cell growth and tumour progression, tumour-associated angiogenesis, migration, invasion, as well as suppression of antitumour immune responses [26, 27]. M-CSF is overexpressed in melanoma cells and increased M-CSF plasma levels are associated with melanoma progression [25]. Furthermore, M-CSF receptor (M-CSFR) expression is elevated in BRAF-mutated melanoma and regulates melanoma growth and invasion [28]. Thus, the combination of BRAFi and M-CSFR blockade enhances T-cell cytotoxicity response and improves the BRAFi antitumour effect in murine melanoma cells [29]. Nevertheless, the contribution of M-CSF to acquired resistance to BRAFi in melanoma involves a complex and context-dependent secretory mechanism that remains poorly understood. Therapeutic manipulation of the secretome is a promising approach in cancer therapy [30, 31]. Therefore, understanding the complex contribution of the secretome to acquired resistance in melanoma may open new avenues to identify novel therapeutic targets in melanoma patients.

Our study aimed to analyse the role of the secretome and specifically of M-CSF in BRAFi-acquired resistance, in metastatic melanoma.

Materials and methods

Cell lines

Eight BRAFV600E-mutant Vemurafenib-sensitive (Vem-S) and resistant (Vem-R) human malignant melanoma cells [9] were used. For cell culture conditions, see Supplemental Information.

Plasma samples from melanoma patient’s collection

Plasma isolation from 35 patients diagnosed with cutaneous BRAF-mutant melanoma seen and treated at the Melanoma Unit of the Hospital Clínic of Barcelona (HCB). Clinical information was retrieved for each patient. The Clinical Research Ethics Committee of the HCB (study registry 2013/8305) approved this study and each study participant signed written informed consent in accordance with the Declaration of Helsinki (see Supplemental Information).

Compounds/drugs

Vemurafenib (PLX4032) (Vem), was purchased from Roche (10123741). Mibefradil (Mib) was acquired from Santa Cruz Biotechnology (204083 A). Chloroquine (CQ) was obtained from Sigma-Aldrich (C6628). The recombinant protein rhM-CSF was purchased from Immunotools® (11343113). M-CSF mAb monoclonal antibody was acquired from Deltaclon (TAB-029ML Lacnotuzumab).

Human cytokine array

Conditioned media (CM) from Vem-S and Vem-R were incubated in Human Cytokine Antibody Array G-Series 3 (RayBiotech) following the manufacturer’s protocol. For detailed protocol see Supplemental Information.

ELISA of M-CSF levels in conditioned media or plasma from melanoma patients

M-CSF secreted cytokine in conditioned media of melanoma cells or plasma samples from melanoma patients was analysed by Human M-CSF SimpleStep ELISA kit (ab245714 Abcam).

Cell viability and apoptosis assays

Melanoma cells were treated with Vemurafenib (1 µM), and/or Mibefradil (5 µM) or Chloroquine (12.5 µM), and/or the M-CSF mAb (20 ng/mL) for 24 h or 48 h. Cell viability and apoptosis were assessed by MTT assay or double-staining AnnexinV-PI, respectively [9].

Migration and invasion assays

Wound healing and Transwell assays were performed as described [6, 32].

Monitoring autophagy flux assay

SK-Mel-28R cells were transfected with tfLC3 plasmid (a chimeric mRFP-GFP tandem fluorescent-tagged LC3B construct) with lipofectamine 2000 (Thermofisher) as described previously [33, 34].

Western blot

Western blotting was performed as described previously [5]. The antibodies used and protocol information are detailed in Supplemental Information.

Subcutaneous tumour xenografts and treatment administration

The animal experimental committee approved animal work procedures (CEEA from University of Lleida). Immunodeficient SCID mice (age 12 weeks; weight 20–25 g) were subcutaneously injected with 2 × 106 SK-Mel-28R cells. Mice were treated randomly with Vemurafenib (50 mg/kg) and Chloroquine (30 mg/kg) daily by oral gavage (o.p) whereas; M-CSF mAb (Lacnotuzumab; 4 mg/kg) was administered intraperitoneally (i.p.) every 3 days (n = 6 each group) (see Supplemental Information).

Immunohistochemistry

Proliferation marker (Ki-67) and vascularisation marker ENG (Agilent) were optimised using the IHC Dako system (Glostrup, Denmark) [35] and the percentage of positive cells was analysed using Qupath [36].

Statistical analysis

Cytokines with Fold-change higher than 1.2 were selected and gene ontology network analysis was performed with ClueGO (Version 2.3.2) and Cytoscape (Version 3.4.0) to analyse citoarray results [37]. Statistical analysis was carried out using GraphPad Prism software (mean ± SEM.; n > 3). Statistical significance was evaluated using Kolmogorov–Smirnov normality test followed by one-way or two-way ANOVA and post hoc Bonferroni, or Mann Whitney tests. The Wilcoxon test was used to evaluate correlation values. Fisher exact tests were used to examine clinical variables. Melanoma overall survival (mOS) and disease-free survival (DFS) curves were calculated using the Kaplan–Meier with a log-rank test. p-values are indicated by asterisks *p < 0.05; **p < 0.01; ***p < 0.001.

Results

Secreted media from Vemurafenib-resistant melanoma cell lines induces a resistance phenotype in sensitive melanoma cells

In order to evaluate the effect of Vemurafenib-resistant (Vem-R) melanoma cells secretome on the behaviour of Vemurafenib sensitive (Vem-S) melanoma cells, we first cultured Vem-S cells with their corresponding resistant conditioned media (CM) (Fig. 1a). We then addressed the effect of BRAFi, Vemurafenib, on cell viability and migration/invasion processes. Treatment with Vemurafenib during 48 h decreased Vem-S cell viability. However, Vemurafenib treatment did not alter Vem-S cells viability and migration/invasion rates when the cells were cultured in Vem-R CM (Fig. 1b–d and Supplementary Fig. 1a, b). To further validate the tumour-resistance role of resistant CM secretome, we used plasma from SCID mice with sensitive and resistant melanoma cells xenografts (Fig. 1e). Importantly, the percentage of cell migration, both in control plasma (SCID with non-tumour) and in Vem-S xenograft mouse plasma, decreased with Vemurafenib treatment. In contrast, maintenance of cell migration rates was observed in Vem-S cells incubated with Vem-R xenograft mouse plasma after Vemurafenib treatment (Fig. 1e). These data suggest a key role of the secretome from Vem-R cells in the acquisition of BRAFi resistance in sensitive melanoma cells.

Fig. 1. Secreted media from Vemurafenib-resistant melanoma cell lines induces a resistance phenotype in sensitive melanoma cells.

Fig. 1

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a Schematic representation of the in vitro assays. b Cell viability assay or c Wound healing of M3 and Sk-Mel-28 cells after being cultivated with their own conditioned media (CM) and from the corresponding resistant CM treated with Vemurafenib (1 μM, 48 h). d Representative images of Hoechst nuclear staining of the M3 cell line (x10 microscopic field) and percentage (%) of invasive cells into the matrigel after transwell assay with CM (Vemurafenib 1 μM, 48 h). e (Left) Schematic representation of the in vivo assay. (Right) Wound-healing assay of M3 cells cultivated with 10% of mice plasma from SCID mice with xenografts of A375, M238, A375R, M238R and Sk-Mel-28R melanoma cells treated with Vemurafenib (1 μM, 48 h). Graphs show mean + SEM. n > 3 independent experiments. Statistical analysis was performed using ANOVA and Bonferroni tests (*p < 0.05; **p < 0.01; ***p < 0.001; n.s., non-significant).

Increased pro-tumour cytokine profile of Vemurafenib-resistant melanoma cell lines

We performed a Human Cytokine Array to understand which cytokines and growth factors were linked to the BRAFi-resistant secretory phenotype (Supplementary Fig. 2a). For resistant CM, ClueGo Gene Ontology analysis revealed a specific network related to Chemokine and Cytokine activity and which induced a signalling pathway via JAK-STAT, a proinflammatory, pro-tumour and prosurvival pathway [38] (Fig. 2a and Supplementary Fig. 2b). In deep analysis, as shown in Fig. 2b (Supplementary Fig. 2c), pro-tumoural cytokines, such as GROα, IL-1α, IL-3, IL-4, IL-8, IL-13, M-CSF and TGF-β, were profusely secreted in Vem-R cells in comparison with Vem-S cells. Likewise, angiogenic factors, like EGF, IGF-1, Oncostatin, Thrombopoietin, VEGF and Leptin, were also highly secreted in Vem-R cells (Fig. 2 and Supplementary Fig. 2d). These data indicate that the cytokine profile should create a favourable environment for the acquisition of BRAFi resistance.

Fig. 2. Cytokine secretome profile of Vemurafenib-resistant melanoma cell lines exhibit an increase in pro-tumour cytokines.

Fig. 2

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a GO analysis based on differentially regulated cytokines on resistant melanoma cells. Each node shows an enriched GO term. Heatmap (OD mean) and cytokine expression graphic of b pro-tumour cytokine profile and c pro-angiogenic cytokine profile. Statistical analysis was performed using the Wilcoxson multiple comparison test.

M-CSF promotes the Vemurafenib-resistance phenotype in BRAFV600E melanoma cells

M-CSF is a secreted cytokine that is overexpressed in many tumours, including melanoma [23] and is involved in several pro-tumour effects such as tumour promotion and progression [26, 39]. The cytoarray analysis showed an increased secretion of M-CSF in Vem-R cells compared to Vem-S cells (Fig. 2b and Supplementary Fig. 2c). In addition, we performed an ELISA assay against M-CSF in the conditioned media of Vem-S and Vem-R cells. We observed that resistant CM presented a significant enhancement of M-CSF secreted in the media (Fig. 3a). Subsequently, we evaluated whether M-CSF secretion mediates BRAFi resistance acquisition using recombinant M-CSF protein (rhM-CSF) at different doses. Obtained data indicated that exposure to rhM-CSF did not induce a variation in Vem-S cell viability at any dose (Supplementary Fig. 3a). Moreover, the results showed that combined treatment (rhM-CSF + Vemurafenib) did not alter cell viability in BRAFV600E melanoma cells and promote their migration rates compared to Vemurafenib monotherapy (Fig. 3b and Supplementary Fig. 3b, c). Subsequently, we aimed to evaluate whether neutralisation of this cytokine, by M-CSF monoclonal antibody (M-CSF mAb, Lacnotuzumab) (Supplementary Fig. 3d), could inhibit resistance acquisition in BRAFV600E melanoma cells. Results showed that Vem-S cell, cultured with CM from Vem-R cells treated with M-CSF mAb (20 ng/mL) in combination with Vemurafenib (Fig. 3c), significantly decreased cell viability (Fig. 3d) while M-CSF mAb in monotherapy and in combination with Vem, inhibited migration rates (Fig. 3e). To reassess our findings, a wound-healing assay showed a decreased migration rate in Vem-S melanoma cells cultured with mouse plasma, from Sk-Mel-28R xenografted mice, when we treated cells with M-CSF mAb (Fig. 3f). Therefore, these results suggest that M-CSF in the secretome of resistant cells, has an important role during Vemurafenib-resistance acquisition in BRAFV600E melanoma cells.

Fig. 3. M-CSF promotes the acquisition of Vemurafenib-resistance in BRAFV600E melanoma cells.

Fig. 3

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a Levels of M-CSF in conditioned media of Vem-S and Vem-R cell lines determined by ELISA assay. b Viability and wound-healing assay of sensitive melanoma cells treated with Vemurafenib (1 μM, 48 h) and/or rhM-CSF (200 ng/mL). c Schematic representation of the in vitro assays. d MTT assay or e wound-healing assay of sensitive M3 and SK-Mel-28 cells after exposure to 48 h of their corresponding resistant CM treated with Vemurafenib (1 μM) and/or M-CSF mAb (20 ng/mL). f Wound-healing assay of Sk-Mel-28 cells cultivated with 10% of mice plasma from SCID mice with Sk-Mel-28R xenografts and treated with Vemurafenib (1 μM, 48 h) and/or M-CSF mAb (20 ng/mL). Graphs show mean + SEM. n = 3 independent experiments. Statistical analysis was performed using the ANOVA test, followed by Bonferroni’s multiple comparisons test (*p < 0.05; **p < 0.01; ***p < 0.001; ns, non-significant).

Autophagy blockers combined with Vemurafenib and M-CSF mAb induce cell death and impair migration of BRAFV600E-resistant melanoma cells

First, we describe that M-CSF mAb did not decrease either cell viability or cell migration in Vem-R cells which were treated alone or in combination with Vemurafenib (Supplementary Fig. 4a, b). These data indicate that M-CSF mAb does not affect established Vemurafenib-resistant melanoma.

In previous results, we described that TTCC Cav3.1 isoform and autophagy are increased in Vem-R melanoma cells and that autophagy flux could be a targetable process to treat melanoma [9]. Following this line of enquiry, first, we monitored autophagy flux after M-CSF mAb treatment. Using mRFP-EGFP-LC3B plasmid, we demonstrated that M-CSF mAb treatment induces an autophagy process in Vem-R cells, as we observed an increase in autophagolysosomes (red dots). However, when we treated cells with a TTCC blocker (Mibefradil) we detected an increase in autophagosomes (green-yellow dots), indicating a blockage of basal autophagy (Fig. 4a) [5, 9]. Moreover, incubation with CQ, an autophagy blocker, for 1 h in M-CSF mAb-treated cells elicited a further increase of LC3II expression (Fig. 4b). In summary, these results indicate that M-CSF mAb promotes autophagy flux in Vem-R melanoma cells.

Fig. 4. Autophagic blockers Mibefradil/Chloroquine, Vemurafenib and M-CSF mAb-combined therapy induce apoptotic cell death and impair migration of BRAFV600E-resistant melanoma cells.

Fig. 4

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a Monitoring autophagic flux by mRFP-EGFP-LC3B assay in resistant melanoma cells after M-CSF mAb, Mibefradil or Vemurafenib treatment. b WB and quantification of LC3II protein levels from Sk-Mel-28R melanoma cell line exposed to CQ for 1 h and M-CSF mAb (20 ng/mL). c Cell viability assay, d apoptotic Annexin V assay and e wound-healing assay of Sk-Mel-28R cells treated with Vemurafenib (1 μM) and/or M-CSF mAb (20 ng/mL) and/or combined with Mibefradil (5 μM) or CQ (12.5 μM). Graphs show mean + SEM. n > 3 independent experiments. Statistical analysis was performed using the ANOVA test, followed by Bonferroni’s multiple comparisons test (*p < 0.05; **p < 0.01; ***p < 0.001; ns, non-significant).

It is well established that autophagy inhibitors induce cell death and reduce the migration/invasion potential of Vem-S and Vem-R BRAFV600E–mutant melanoma cells. Furthermore, combination of autophagy blockers with Vemurafenib does not promote synergistic effects in either in vitro culture nor in xenograft mice models [6, 9]. Owing to the potential therapeutic role of M-CSF mAb during the onset of drug resistance, we sought to ascertain whether Vem-R cells were more sensitive to triple treatment using autophagy blocker (Mibefradil or CQ), Vemurafenib and M-CSF mAb compared to monotherapy. As shown in Fig. 4c–e, triple-combined therapy caused a significant reduction of cell viability and migration rates in Vem-R cells compared to TTCC or autophagy blocker (Mibefradil; 5 µM or CQ; 12,5 µM) in monotherapy (Supplementary Fig. 4c, d). Likewise, we observed a significant induction of apoptosis in Vem-R cells after triple therapy (Fig. 4d). In conclusion these results describe that triple-combined treatment induces apoptosis and reduces migration rates in Vem-R melanoma cells.

Chloroquine, Vemurafenib and M-CSF mAb-combined therapy reduces tumour growth in vivo

To confirm our in vitro results, we subcutaneously injected resistant melanoma cells (Sk-mel-28R) into SCID mice and treated them for 2 weeks (n = 6) (Fig. 5a). Subcutaneous tumours treated with triple-combined therapy demonstrated slower growth compared to those treated with CQ, which grew similarly to Vemurafenib or Vemurafenib+M-CSF mAb-treated tumours (Fig. 5b). Moreover, with the latter we observed that mice lose weight during treatment administration whereas triple-combined therapy presents a milder decrease in body weight, indicating increased well-being (Fig. 5c). Furthermore, tumours treated with triple combination show fewer Ki-67 and ENG-positive cells, indicating a reduction of cell proliferation rates and vascularisation in the xenografted tumour, respectively (Fig. 5d, e). Altogether, these data suggest that autophagy modulation by triple-combined treatment significantly reduces tumour growth in Vem-R melanoma cells due to autophagy blockade, showing a synergistic effect.

Fig. 5. Autophagic blockers Mibefradil/Chloroquine, Vemurafenib and M-CSF mAb-combined therapy reduce tumour growth and vascularisation of BRAFV600E-resistant melanoma cells.

Fig. 5

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a Workflow of the in vivo experiment. (o.p. = orally; i.p. = intraperitoneal). b Tumour growth curves under treatment with Vemurafenib (50 mg/kg), CQ (30 mg/kg), M-CSF mAb (Lacnotuzumab) (4 mg/kg) or the combination therapy for 2 weeks in mice with SK-Mel-28R xenografts (n = 6). c Weight monitoring during all the following treatments. d Ki-67 Immunochemistry detection in xenografts (left) % of positive Ki-67 (right) and representative images of the Ki-67 immunostaining. e (left) % of positive immunostaining of ENG and (right) images of vascularisation represented by ENG immunostaining. Statistical analysis was performed using the ANOVA test, followed by Bonferroni’s multiple comparisons test (*p < 0.05; **p < 0.01; ***p < 0.001; ns, non-significant).

M-CSF plasma levels correlate with melanoma prognosis in patients

We assembled a cohort of 35 patients with BRAF-mutant melanoma and collected plasma before (n = 18) or closely after metastasis diagnosis (n = 17) (Fig. 6a). Clinical data were gathered for each melanoma patient (Fig. 6b and Supplementary Fig. 5a). We evaluated M-CSF plasma levels by ELISA assay. Using the median value as optimal cut-off, we divided the levels into high (>5 ng/mL) or low (≤5 ng/mL) M-CSF groups (Supplementary Fig. 5b). First, we observed that there is no significant correlation between M-CSF levels with melanoma-specific overall survival (mOS) and the disease-free survival (DFS) of patients with melanoma (Supplementary Fig. 5c, d). We went on to evaluate whether the timing of plasma collection (before or after metastatic development) was important in determining whether M-CSF levels could be a good prognostic biomarker. First, we selected plasma samples before metastasis (n = 18), where no significant differences were observed between M-CSF plasma levels and prognostic values (survival and development of metastasis) in primary melanoma patients (Supplementary Fig. 6a). Subsequently, we selected samples from patients after metastasis diagnosis and before treatment initiation (n = 17). Interestingly, we observed that metastatic patients with high M-CSF levels (>5 ng/mL) had worse mOS compared to patients with low M-CSF levels (≤5 ng/mL) (p = 0.0175) (Fig. 6c). Furthermore, all patients with metastatic melanoma who eventually died from it presented high M-CSF levels in the plasma sample collected before treatment (Fig. 6d, e).

Fig. 6. M-CSF plasma levels determine melanoma OS and DFS in metastatic melanoma patients.

Fig. 6

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a Timeline of plasma collection and patient treatment. b Clinical parameters from 35 patients with BRAFV600E advanced-stage melanoma. MAP kinase-targeted therapy: BRAFi (Vemurafenib, Dabrafenib or LGX818) in monotherapy or in combination with MEKi (Dabrafenib/Trametinib, LGX818/MEK162, Vemurafenib/Cobimetinib). Response to the treatment: complete response (CR), partial responders (PR), and non-responders (stable disease (SD) and progressive disease (PD)). c Kaplan–Meier of melanoma overall survival (mOS) curve of metastatic melanoma patients (n = 17) with <5 ng/mL of M-CSF vs. >5 ng/mL. d Contingency of alive/dead patients depending on their M-CSF levels and e analysis of M-CSF plasma levels depending on the status of the patients (alive/dead). f Kaplan–Meier of melanoma disease-free survival (%DFS) of metastatic melanoma patients with <5 ng/mL vs. > 5 ng/mL of M-CSF plasma levels. g M-CSF levels depending on time to relapse after treatment (<12 vs. >12 months). Fisher exact tests were used to examine clinical variables and the ANOVA test for the statistical analysis of M-CSF plasma levels. Melanoma overall survival (mOS) and disease-free survival (DFS) curves were calculated using the Kaplan–Meier method with a log-rank test. p-values are indicated by asterisks *p < 0.05; **p < 0.01; ***p < 0.001.

Melanoma patients included in our cohort were treated with either a combination of MAP kinase-targeted therapy (BRAFi + MEKi such as Dabrafenib/Trametinib, LGX818/MEK162, Vemurafenib/Cobimetinib) or with BRAFi monotherapy (Vemurafenib, Dabrafenib or LGX818) (Fig. 6b). Metastatic melanoma patients with lower M-CSF levels presented better DFS (from treatment initiation to relapse) compared to patients with higher M-CSF levels (p = 0.0022) (Fig. 6f). Overall, drug resistance (measured as relapse occurrence) appeared at least 12 months after the start of each treatment for almost all patients with lower M-CSF levels (Fig. 6g). During treatment, patients were classified according to their best response to the treatment: complete response (CR), partial responders (PR), and non-responders (stable disease (SD) and progressive disease (PD)) (Fig. 6b). We observed that all metastatic melanoma patients with CR (3/17; 17.6%) had low levels of M-CSF ( < 5 ng/mL) while all patients who did not respond to treatment (PD) (2/17; 11.8%) had high levels of M-CSF (Supplementary Fig. 6b).

Next, according to specific therapy, the association of higher mOS and DFS with low M-CSF levels was maintained in patients treated with combined therapy (BRAFi + MEKi) (Supplementary Fig. 6c, d). Owing to the low number of melanoma patients treated with BRAFi monotherapy, we did not observe a significant association of M-CSF levels in this sub-cohort (Supplementary Fig. 6e). Overall, these findings suggest that low levels of M-CSF are associated with better prognosis and disease progression in patients with BRAF–mutated metastatic melanoma.

Discussion

New approaches to cancer research evaluate tumour cell secretomes and their environment, which influences tumour biological properties, including metastatic potential and resistance to therapy [20, 40–43]. Therefore, understanding the secretome and identifying suitable tumour targets could help design powerful therapies, capable of triggering regression of tumour loads and achieving the desired outcome [20, 43, 44].

The use of CM of tumour cells is a promising method [19] to describe the secretome of BRAFV600E sensitive melanoma and resistant melanoma cells, and to distinguish them from one another. In the present study, we observed that the secretome of resistant melanoma cells is complex and rich in pro-tumoural cytokines such as GRO-α, IL-1α, IL-3, IL-4, IL-5, IL-6, IL-10, IL-13, M-CSF, and TGF-β1, which affect tumour progression [45]. Furthermore, and the secretome of resistant melanoma cells shows a greater number of angiogenic factors like EGF, VEGF and IGF-I, which are needed to promote the establishment of metastatic colonies [46]. We have for the first time described an increase in M-CSF secretion in BRAFV600E-mutant-resistant melanoma cells, which can induce BRAFi-resistant phenotype in sensitive cells.

Most of the monoclonal antibodies usually employed against M-CSF blocked its receptor, which reduces tumour growth in prostate cancer, osteosarcoma, lymphoma, breast cancer metastasis, glioblastoma and melanoma [47–49]. Moreover, exist several ongoing clinical trials test M-CSF mAb or M-CSFR inhibitors in combination with chemotherapy, BRAF/MEK inhibitors or immunosuppressive therapies in advanced malignancies, including melanoma [31, 49]. In this work, we used an antibody against M-CSF (M-CSF mAb-Lacnotuzumab) as an antitumour therapy for resistant melanoma cells. BRAFi shows high initial antitumour effectiveness, but limited response due to resistance acquisition in BRAFV600E-mutated melanoma patients [2, 4, 18, 50, 51]. Moreover, multiple reports describe that targeting M-CSF receptor (PLX3397) increases the antitumour effect of BRAFi in melanoma preclinical and clinical models [29, 31, 52]. We demonstrated that while M-CSF mAb did not modulate the behaviour of Vem-R cells, it was able to reduce migration and induce apoptosis of parental cells treated with CM of Vem-R cells. Manipulation of the secretoma in BRAFV600E melanoma cells could be a promising approach to treat melanoma patients [30, 31].

As previously described, once melanoma cells become resistant to BRAFi, autophagy blockers induce apoptosis and impair migration in Vem-R cells [9]. Based on this data, triple combination (autophagy blocker + Vem + M-CSF mAb) induced apoptosis, impaired migration and reduced tumour growth in vivo compared to monotherapy treatment. All the data support the idea that autophagy inhibition along with M-CSF mAb, as a therapy-inhibiting tumour secretome, may offer a promising new strategy to deal with resistant mechanisms in melanoma cells [9, 10, 18, 51].

Finally, we determined for the first time that high secreted M-CSF plasma levels correlate with worse prognosis (melanoma OS and DFS) in BRAFV600E metastatic melanoma patients. M-CSF expression has been associated with worse prognoses in several cancer types, such as prostatic, breast and renal carcinomas. This fact has been explained by the effect on tumour-associated macrophages (TAMs), which could act as immunosuppressive tumour growth promoters and enhancers of resistance acquisition to therapy [25, 28, 53, 54]. Neubert and co-workers showed a progressive increase of M-CSF levels in the peripheral blood of melanoma patients, indicating that M-CSF secretion increases during tumour development [25]. Currently, ongoing clinical trials are evaluating the combination of M-CSF mAb or M-CSFR mAb therapy with BRAF/MEK inhibitors (Dabrafenib/Trametinib or Vemurafenib/Cobimetinib) in melanoma patients to determine whether it helps in overcoming the resistance observed in conventional treatments (NCT03455764 or NCT03101254) (https://clinicaltrials.gov). According to our results, analysis of M-CSF plasma levels could be a useful strategy to predict prognosis and response to MAP kinase-targeted therapy in metastatic melanoma patients. In light of these findings, our data suggest that M-CSF is crucial during the acquisition of BRAFi resistance in melanoma cells. Therefore, M-CSF blockade, as a therapy-inhibiting tumour secretome, could be key to preventing the rapid emergence of resistant mechanism and lead to a better prognosis for melanoma patients.

Supplementary information

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Supplementary Figures (1.1MB, pdf)

Acknowledgements

This work was supported by grants from ISCIII/FEDER “Una manera de hacer Europa” (PI1500711 to R.M.M. & PI18/00573 to RMM & AM and PI20/00502 to NE) and CIBERONC-CB16/12/00231 to XMG, Fundació la Marató de TV3 (FMTV 201331-31) to RM and Generalitat de Catalunya (2014/SGR138) to XMG. CB and PS hold a predoctoral fellowship from UdL-IRBLleida. IR holds a predoctoral fellowship from AECC. AM holds a postdoctoral fellowship from AECC. The cell culture experiments were performed in the Cell Culture Scientific & Technical Service from Universitat de Lleida (UdL), Lleida, Spain. Work supported by the Xarxa de Bancs de Tumours de Catalunya sponsored by Pla Director d’Oncología de Catalunya (XBTC)”, IRBLleida Biobank (B.0000682) and PLATAFORMA BIOBANCOS (PT17/0015/0027; PT20/00021) and HCB-IDIBAPS Biobank (R120904-090) integrated in the Spanish National Biobank Network (ISCIII Code C 0.000.334). The research at the Melanoma Unit from Hospital Clinic of Barcelona was partially funded by Insituto de Salud Carlos III (ISCIII), Spain, through projects PI18/00419 and PI18/01077, and co-funded by the European Union; by the grant AC16/00081, integrated in the Plan Estatal I + D + I, IMMUSPHINX-Transcan-2; by the CIBER de Enfermedades Raras of ISCIII, Spain, cofinanced by European Development Regional Fund “A way to achieve Europe” ERDF; and by the Generalitat de Catalunya (AGAUR 2017/SGR1134 and CERCA Program). We are grateful to our patients and relatives, to physicians and nurses from the Melanoma Unit of Hospital Clínic of Barcelona for collecting patients samples and data, and to Judit Mateu from the “Melanoma: image, genetics and immunology” group at IDIBAPS for her technical assistance. NCL holds a predoctoral fellowship from Ministerio de Educación, Cultura y Deportes, Spain (FPU17/05453).

Author contributions

Conceptualisation: CB, PS, RM, AM; Data curation: CB, PS, IR, CM, RN, OM, IU, NE, XS, AM; Formal analysis: CB, PS, OM, AM; Funding acquisition: RM, AM, SP, XMG, NE; Investigation: CB, PS, IR, CM, RN, OM, IU, NE, MP, NCLl, AM. Methodology: CB, PS, OM, AM. Project administration: CB, PS, AM. Resources: SP, MP, NCLl, XMG, NE, RM, AM; Software: CB, PS, OM. Supervision: RM, AM; Validation: CB, PS; Visualisation: CB, PS, AM; Writing—original draft preparation: CB, PS, AM; Writing—review and editing: CB, PS, OM, SP, RM, AM.

Data availability

Not applicable

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

The animal experimental committee approved animal work procedures (CEEA from University of Lleida). The Clinical Research Ethics Committee of the HCB (study registry 2013/8305) approved this study and each study participant signed written informed consent in accordance with the Declaration of Helsinki.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: C. Barceló, P. Sisó.

These authors jointly supervised this work: R. M. Martí, A. Macià.

Contributor Information

R. M. Martí, Email: marti@medicina.udl.cat

A. Macià, Email: amacia@irblleida.cat

Supplementary information

The online version contains supplementary material available at 10.1038/s41416-022-01886-4.

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Supplementary Materials

Supplementary Information (23KB, docx)
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