Therapy-induced tumour secretomes promote resistance and tumour progression

Abstract

Drug resistance invariably limits the clinical efficacy of targeted therapy with kinase inhibitors against cancer. Here we show that targeted therapy with BRAF, ALK or EGFR kinase inhibitors induces a complex network of secreted signals in drug-stressed human and mouse melanoma and human lung adenocarcinoma cells. This therapy-induced secretome stimulates the outgrowth, dissemination and metastasis of drug-resistant cancer cell clones and supports the survival of drug-sensitive cancer cells, contributing to incomplete tumour regression. The tumour-promoting secretome of melanoma cells treated with the kinase inhibitor vemurafenib is driven by downregulation of the transcription factor FRA1. In situ transcriptome analysis of drug-resistant melanoma cells responding to the regressing tumour microenvironment revealed hyperactivation of several signalling pathways, most prominently the AKT pathway. Dual inhibition of RAF and the PI(3)K/AKT/mTOR intracellular signalling pathways blunted the outgrowth of the drug-resistant cell population in BRAF mutant human melanoma, suggesting this combination therapy as a strategy against tumour relapse. Thus, therapeutic inhibition of oncogenic drivers induces vast secretome changes in drug-sensitive cancer cells, paradoxically establishing a tumour microenvironment that supports the expansion of drug-resistant clones, but is susceptible to combination therapy.

Extended Data Figure 1. Targeted therapy or oncogene knockdown leads to regression of sensitive melanoma and lung adenocarcinoma tumours but accelerates the proliferation and seeding of residual drug-resistant cells in vivo

a, FACS analysis of sensitive A375 and vemurafenib-resistant A375^R cells expressing TK-GFP-Luciferase (TGL), at tumour implantation and after two weeks at start of therapy (n = 8 tumours) Plots depict representative images. b, Tumour volume of A375 cells treated with vehicle or vemurafenib over time (vehicle, n = 8; vemurafenib, n = 12 tumours). c, Representative sections of A375/A375^R-TGL tumours at 0, 1, 3, and 6 days of vemurafenib treatment analysed with immunofluorescence (IF) against GFP. Arrows indicate emerging clusters of GFP+ resistant cells. Scale bar: 2mm. d, Quantification of BrdU incorporation into vemurafenib-resistant A375^R-TGL cells in A375/A375^R tumours treated with vehicle or vemurafenib for 6 days (vehicle, n = 13 fields of vision (FOV) of 3 tumours; vemurafenib n = 18 FOV of 4 tumours). e, Fold change of photon flux of TGL-expressing A375^R cells in A375 tumours or A375^R tumours alone treated with vehicle or dabrafenib for 8 days (A375/A375^R: vehicle, n = 15; dabrafenib, n = 14; A375^R: vehicle, n = 8; dabrafenib, n = 7 tumours). f, Tumour volume of doxycycline-inducible BRAF knockdown A375-i-shBRAF-derived xenograft tumours treated with vehicle or doxycycline over time (vehicle, n = 5; doxycycline, n = 4 tumours). g, Photon flux of TGL-expressing A375^R cells mixed in A375-i-shBRAF tumours treated with vehicle or doxycycline (vehicle, n = 10; doxycycline, n = 11 tumours). h, Fold change of photon flux of TGL-expressing vemurafenib-resistant M249^R4 tumours treated with vehicle or vemurafenib (n = 16 tumours). i–k. Co-implantation assay of tumours treated with vehicle or corresponding targeted therapy with BLI quantification after 5–8 days. i, Fold change of photon flux of TGL-expressing vemurafenib-resistant YUMM1.7^R cells mixed in unlabelled, vemurafenib-sensitive YUMM1.7 tumours or YUMM1.7^R tumours alone (YUMM1.7/YUMM1.7^R: n = 24; YUMM1.7^R: n = 20 tumours ). j, Fold change of photon flux of TGL-expressing, intrinsically vemurafenib resistant B16 cells mixed in vemurafenib-sensitive YUMM1.1 tumours or B16 tumours alone (YUMM1.1/B16: vehicle, n = 12; vemurafenib, n = 16; B16: n = 20 tumours) k, A375^R mixed in crizotinib-sensitive H3122 cells or A375^R tumours alone (H3122/A375^R: vehicle, n = 14; crizotinib, n = 13; A375^R: n = 12 tumours). l, Photon flux of tumours established from intrinsically resistant drug resistant cells alone, treated with vehicle, crizotinib or erlotinib (crizotinib resistant PC9, H2030 or erlotinib resistant A375^R) (n (from left to right on the graph, in this order) = 12, 12, 7, 12, 16, 16 tumours, respectively). m, Summary table of the model systems and conditions used in vivo. n, On the left, representative IF images of vemurafenib treated, sensitive tumours 7h or 5d after intracardiac injection with A375^R-TGL cells; sections stained for GFP (A375^R, green), collagen type IV (blood vessels, red), and DAPI (nuclei, blue). On the right, quantification of A375^R single cells and cell clusters (≥2 cells) infiltrating an A375 tumour treated with vehicle or vemurafenib after intracardiac injection of A375^R cells (GFP+ cells were scored in at least 10 whole sections of at least 4 tumours). Data in b, e–l, n are presented as averages, error bars represent s.e.m., in f, center line is median, whiskers are min to max. P values shown were calculated by a two-tailed Mann-Whitney test (n.s.=not significant).

Extended Data Figure 2. The secretome of vemurafenib-treated melanoma and crizotinib- or erlotinib-treated lung adenocarcinoma cells stimulates the proliferation and migration of drug resistant cells in vitro and occurs prior to apoptosis and senescence

a, Quantification of the co-culture assay, depicted in Fig. 2a, 7 days after addition of resistant A375^R-TGL cells (n = 4 biological replicates). P values were calculated using a Student’s t-test. b, c, Drug sensitive cells were pre-treated with vehicle or drug (crizotinib or erlotinib) for 48h before 5×10² TGL-expressing, drug-resistant cells were added. Growth was monitored by BLI and quantified 7 days after addition of the resistant cell population, (n = 8 biological replicates), P values were calculated using a Student’s t-test. b, Quantification and representative images of TGL-expressing H2030 cells alone or co-cultured with crizotinib sensitive H3122 cells and treated with vehicle or crizotinib c, Quantification and representative images of TGL-expressing A375^R cells alone or co-cultured with erlotinib sensitive HCC827 cells and treated with vehicle or erlotinib. d, Relative number of vemurafenib-resistant LOX^R cells after 3 days in the presence of CM derived from A375 and UACC62 cells (n = 3 biological replicates). e, Representative IF for Ki67 in drug-resistant YUMM1.7^R cells cultured in CM from YUMM1.7 cells. f, Relative number of vemurafenib-resistant melanoma cells with different, clinically relevant resistance mechanisms after 3 days in the presence of CM derived from A375 cells. SKMEL239#3 expressing the p61 BRAFV600E splice variant, A375 expressing NRAS^Q61K or the constitutively active MEK variant MEK-DD (n = 5 biological replicates). g, Relative cell number of intrinsically vemurafenib resistant lung adenocarcioma cells (H2030, PC9) or crizotinib and erlotinib resistant melanoma cells (A375^R) after 3 days cultured in the presence of CM from vemurafenib-treated melanoma or crizotinib- and erlotinib-treated lung adenocarcinoma (n = 6 in all, except for A375^R with HCC827-CM, n = 4 biological replicates). h, Representative image of A375^R cells migrated towards A375-derived CM-vehicle or CM-vemurafenib. i, Relative migration towards CM from different sources and different resistant test cells as indicated (n = 10 FOV). P values were calculated using a two-tailed Mann-Whitney test (** p<0.01, **** p<0.0001). j, Representative graph and quantification of real-time migration of A375^R cells in the presence of CM derived from A375 cells as measured by the xCELLigence System (n = 4 biological replicates). P-value shown was calculated using a two-tailed Mann-Whitney test. k, Monolayer gap closing assay of A375^R cells in the presence of CM derived from A375 cells with representative light microscopy images and quantification of gap closure over time. l, Immunoblotting for cleaved caspase-3 and phosphorylated ERK protein levels in vemurafenib-sensitive melanoma cell lines after 72h of vemurafenib treatment. m, β-galactosidase staining of A375 cells treated with vemurafenib for 72h or 8 days. Data are presented as averages, error bars represent s.e.m.

Extended Data Figure 3. The therapy-induced secretome of sensitive cells overlaps significantly in melanoma and lung adenocarcinoma cells and appears after gene expression changes enriched for transcriptional regulators

a–b, GO analysis (revigo.irb.hr) of gene expression changes after 6h of vemurafenib treatment of A375 cells with (a) spatial representation of enriched GO terms and (b) the molecular functions significantly affected. c, Heat map, representing the expression levels of commonly up- and down-regulated genes in vemurafenib-treated A375-derived xenograft tumours (5 days) and A375 cells in vitro (48h). d, Principal component analysis of vemurafenib-sensitive Colo800 and UACC62 melanoma cells and crizotinib-sensitive H3122 lung adenocarcinoma cells treated in vitro with vehicle or vemurafenib or crizotinib for 48h. e, Venn Diagram indicating the overlap of genes in the extracellular region (GO:0005576) up-regulated after 48h of vemurafenib treatment in A375, Colo800, and UACC62 melanoma cell lines. f, Venn Diagram indicating the overlap of genes in the extracellular region (GO:0005576) up-regulated after 48h of vemurafenib treatment in at least 2/3 melanoma models and after 48h of crizotinib treatment in the H3122 lung adenocarcinoma cell line. g, Venn Diagram indicating the overlap of genes in the extracellular region (GO:0005576) down-regulated after 48h of vemurafenib treatment in A375, Colo800, and UACC62 melanoma cell lines. f, Venn Diagram indicating the overlap of genes in the extracellular region (GO:0005576) up-regulated after 48h of vemurafenib treatment in at least 2/3 melanoma models and after 48h of crizotinib treatment in the H3122 lung adenocarcinoma cell line. P values shown were calculated using a Hypergeometric probability test.

Extended Data Figure 4. Vemurafenib treatment induces widespread changes in the intra-tumour immune cell composition and stromal cytokine composition in tumours regressing during targeted therapy

a–b, Fluorescence-activated cell sorting (FACS) analysis of murine immune cell populations in A375-derived xenograft tumours treated with vehicle or vemurafenib for 5 days. a, Representative image and b, quantification of intra-tumour composition of indicated immune cell populations (vehicle, n = 4; vemurafenib, n = 6 tumours). c–d, Cytokine array of murine stroma-derived cytokines within A375-derived xenograft tumours treated with vehicle or vemurafenib for 5 days. c, Representative image and d, quantification of down- and up-regulated cytokines (n = 4 tumours). P values shown were calculated by a two-tailed Mann-Whitney test. Data are averages, error bars represent s.e.m.

Extended Data Figure 5. Targeted therapy induces down-regulation of FRA1 in drug-sensitive tumour cells

a, List of transcription factors predicted to regulate the vemurafenib-induced reactive secretome in A375 cells, and a heat map of the corresponding transcription factor gene expression levels in these cells. Red represents high, yellow medium and blue low relative expression on the colour scale. b, Immunoblotting of phosphorylated and total FRA1 protein levels in A375 and YUMM1.7 melanoma cell lines treated with vemurafenib for 24h. c, Relative mRNA levels of FRA1 in H3122 cells treated with crizotinib (500nM) and HCC827 treated with erlotinib (10nM) at different time points (n = 4 technical replicates) d, Relative mRNA levels of FRA1 in A375^R cells treated with vemurafenib at different time points (n = 4 technical replicates). e, Immunofluorescence staining of FRA1 (red) and DAPI (blue) in biopsies from melanoma patients before and after vemurafenib treatment (clinical information can be found in Extended Data Table 1).

Extended Data Figure 6. The secretome of melanoma cells with FRA1 knockdown stimulates proliferation and migration of A375^R cells in vitro and in vivo

a, Immunoblotting of phosphorylated and total FRA1 protein levels in A375 cells transduced with control shRNA, with or without additional vemurafenib treatment, or shRNAs targeting FRA1. b, Photon flux and representative BLI images of TGL-expressing A375^R cells co-cultured with A375 cells expressing control shRNA (with or without vemurafenib treatment) or FRA1-targeting shRNAs after 7 days (n = 9 biological replicates). c, Relative number of A375^R cells after 3 days in the presence of CM derived from A375 cells transduced with control shRNA, with or without additional vemurafenib treatment, or FRA1 shRNAs (n = 3 biological replicates). d, Migration of A375^R cells towards CM derived from A375 cells transduced with control shRNA (with or without vemurafenib treatment) or FRA1 shRNAs using a Boyden Chamber Assay (shCTRL, n = 15; all other groups n = 10 FOV) e, Relative mRNA levels of selected secreted factors and TF’s of A375 cells expressing control (shCTRL) or a hairpin targeting FRA1 (shFRA1#1), treated with vehicle or vemurafenib (24h). f, Bioluminescent signal of A375^R-TGL cells 8 days after subcutaneous co-implantation with UACC62 cells expressing a control or a short hairpin for FRA1 (shCTRL, n = 12; shFRA1, n = 20 tumours). Data are averages, error bars represent s.e.m. P values were calculated by Student’s t-test, ***p<0.01, ***p<0.001, ****p<0.0001.

Extended Data Figure 7. The therapy-induced secretome (TIS) includes up-regulated positive regulators and a loss of negative regulators of the PI3K/AKT/mTOR pathway, which is activated in sensitive and resistant cells in vitro and in vivo

a, Enriched biological processes and b, inferred drug vulnerabilities as determined by Ingenuity pathway analysis of gene expression data from vemurafenib-resistant A375^R cells responding to signals from the reactive tumour microenvironment of a tumour regressing during targeted therapy in vivo (for experimental setup see Fig. 1a and the methods section). c, Left, immunoblotting of phosphorylated AKT^S473 and phosphorylated ERK protein levels in A375 cells treated with vehicle or vemurafenib at different time points during the generation of conditioned media (CM). Right, immunoblotting of phosphorylated AKT^S473 and phosphorylated ERK protein levels in A375 cells after short-term exposure to CM derived from A375 cells treated with vehicle or vemurafenib. d, Immunoblotting of phosphorylated AKT^S473 and phosphorylated FRA1 protein levels in A375-derived xenograft tumours treated with vehicle or vemurafenib for 5 days. Normalized quantification of phospho-AKT^S473/tubulin in the bottom panel. e, Immunoblotting of a range of pathways nodes in A375^R cells treated with CM-vehicle or CM-vemurafenib, derived from A375 cells, for 15, 30, 60, or 120min. f, Cancer cell-derived IGFBP3 levels (left) and murine stromal IGF1 levels (right) in A375-derived xenograft tumours treated with vehicle or vemurafenib for 5 days as determined by ELISA (n = 4 tumours) g, Cancer cell-derived IGFBP3 levels in CM from indicated melanoma cell lines treated with vehicle or vemurafenib as determined by ELISA (n = 3 technical replicates of at least 2 CM). h, IGFBP3 levels in CM derived from A375 cells expressing control or shRNAs targeting IGFBP3 as determined by ELISA (n = 3 technical replicates). i, Immunoblotting of phosphorylated AKT^S473 in A375^R cells after incubation with CM of A375 cells expressing control or a short hairpin targeting IGFBP3. j, Phosphorylation status of AKT^S473 in A375^R cells after incubation for 15 min with CM, IGF1, and IGFBP3 as indicated. k, Bioluminescent signal of A375^R-TGL cells 10 days after co-implantation with A375 cells expressing a control hairpin (shCTRL) or a hairpin targeting IGFBP3 (shIGFBP3#1) (n = 10 tumours). P values shown were calculated by a two-tailed Mann-Whitney test. Data are averages, error bars represent s.e.m.

Extended Data Figure 8. Dual inhibition of RAF and the AKT/mTOR pathway blunts the effects of the regressing tumour environment on the resistant cell population

a, Relative photon flux and representative BLI images of GFP/luciferase expressing A375^R cells co-cultured with A375 cells and treated with vehicle, vemurafenib, or the combination of vemurafenib and either MK2206 (AKTi, 2μM) or BEZ235 (PI3K/mTORi, 300nM) for 7 days (n = 2–3 biological replicates). b, Relative number of A375^R cells after 3 days in the presence of CM-vehicle or CM-vemurafenib with additional BEZ235 (300nM) (n = 3 biological replicates). c, Mice bearing tumours consisting of A375/A375^R cells or A375^R cells alone were treated with drugs as indicated. Bioluminescent signal of TGL-expressing A375^R cells was determined on day 5 of treatment (n = 16, 16, 12, 12, 12, 16 tumours, respectively). d, Mice bearing tumours consisting of unlabelled A375 cells were pre-treated for 3 days with drugs as indicated and 1×10⁵ TGL-expressing A375^R cells were inoculated in the arterial circulation. Drug treatment was continued and seeding of resistant cells to the primary tumour was quantified by BLI. Representative BLI images on the right (vehicle, n = 4; vemurafenib n = 10, vemurafenib+BEZ235, n = 10 tumours). P values were calculated by a two-tailed Mann-Whitney test. Data are averages, error bars represent s.e.m.

Extended Data Figure 9. Characterization of cell lines in response to targeted therapy

a–h, Relative survival of human melanoma cell lines (A375, Colo800, UACC62) (a,c,e), and the murine melanoma cell line YUMM1.7 (g) and corresponding vemurafenib-resistant derivatives (A375^R, Colo800^R, UACC62^R, YUMM1.7^R) under increasing concentrations of vemurafenib. Immunoblotting of phosphorylated ERK protein levels in indicated melanoma cell lines in the presence of increasing concentrations of vemurafenib (b,d,f,h). i, Immunoblotting of protein levels of MET, EGFR, BRAF, PDGFRβ, phosphorylated AKT, and phosphorylated ERK in vemurafenib-sensitive and -resistant pairs of human melanoma cell lines (A375, Colo800, UACC62). j, Immunoblotting of phosphorylated ERK and phosphorylated AKT^S473 protein levels in HCC827 lung adenocarcinoma cells in the presence of increasing concentrations of erlotinib. k, Immunoblotting of phosphorylated ERK protein levels in H3122 lung adenocarcinoma cells in the presence of increasing concentrations of crizotinib.

Figure 1. The regressing tumour microenvironment stimulates the outgrowth, infiltration and metastasis of drug-resistant clones

a, Schematic of the experimental setup. b, Bioluminescent signal of drug-resistant A375^R-TGL cells in vemurafenib-sensitive, A375 tumours, treated with vehicle or vemurafenib for 5 days (vehicle, n = 36; vemurafenib, n = 15 tumours). c, EdU incorporation in A375^R-TGL cells in A375/A375^R-TGL tumours treated with vehicle or vemurafenib for 4 days, as determined by FACS (vehicle, n = 8; vemurafenib, n = 6 tumours). d, Bioluminescent signal of A375^R-TGL tumours alone, treated with vehicle or vemurafenib for 5 days (vehicle, n = 38; vemurafenib, n = 15 tumours). e, Bioluminescent signal of TGL-expressing drug-resistant cancer cells (A375^R, M249^R4, PC9, H2030) in drug-sensitive tumours (Colo800, LOX, UACC62, M249, H3122, HCC827) treated with vehicle or drugs (vemurafenib, crizotinib, erlotinib) for 5 days (n (from left to right on the graph, in this order) = 6, 7, 12, 12, 9, 9, 25, 26, 9, 12, 12, 12, 16, 11 tumours). f, Spontaneous lung metastasis by A375^R cells in mice bearing A375/A375^R-TGL tumours treated with vehicle or vemurafenib (10 days), visualized by BLI (n = 4). g, Seeding of A375^R-TGL cells from the circulation to unlabelled, subcutaneous A375 tumours of mice treated with vehicle or vemurafenib. Signal in the tumour was quantified by BLI (vehicle, n = 30; vemurafenib, n = 34 tumours; three independent experiments combined). h, Treatment response, determined by tumour size, of subcutaneous A375 tumours allowed to be seeded by A375^R−TGL cells from the circulation or mock injected (vehicle, n = 16; vemurafenib, n = 8 tumours). Data in b–e,g,h, are average; error bars represent s.e.m; data in f, center line is median, whiskers are min. to max. P values shown were calculated using a two-tailed Mann-Whitney test (* p<0.05, ** p<0.01, *** p<0.001, n.s.= not significant).

Figure 2. The secretome of RAF and ALK inhibitor treated tumour cells increases proliferation and migration of drug-resistant cells and supports the survival of drug-sensitive cells

a, Schematic (left) and representative BLI images (right) after 7 days of co-culture. Average fold change (FC) of BLI signal from A375^R-TGL cells in vemurafenib treated wells relative to vehicle treated control wells is depicted on the right (n = 4 biological replicates). b, c, Conditioned media (CM) was derived from drug-sensitive cells, treated with vehicle, vemurafenib, or crizotinib. Drug resistant cells were grown in this CM and the cell number was determined on day 3. Drug-sensitive and drug-resistant cell lines and drugs used to generate CM as indicated. b, (n = 3 biological replicates). c, (n = 6 biological replicates). d, Schematic diagram of the migration assay (upper panel) and relative migration of A375^R cells towards CM from different sources as indicated (lower panel, n = 10 FOV). P values were calculated using a two-tailed Mann-Whitney test (**** p<0.0001). e, Survival assay of drug-sensitive A375 cells cultured in CM and treated with vemurafenib, assessed on day 3(n = 3 biological replicates). f, Apoptosis rate of A375 cells cultured in CM and treated with vemurafenib (3μM) (n = 3 biological replicates). Data are presented as average; error bars represent s.e.m.

Figure 3. FRA1 down-regulation during RAFi treatment drives the reactive secretome

a, Principal component analysis of drug-sensitive A375 cells treated in vitro with vehicle or vemurafenib for 6h or 48h. b, Volcano plots show genes significantly deregulated by vemurafenib treatment after 6h (left) or 48h (right). Transcription factors (TF) and gene products in the extracellular region are depicted in green (down-regulated) and red (up-regulated) (n = 3 tumours) c, Relative mRNA levels of FRA1 during vemurafenib exposure [0.1–1 μM]. d, Representative IF staining of A375/A375^R tumours for GFP (A375^R, green) and FRA1 (red) after vehicle or vemurafenib treatment (5 days). e, Representative IF staining for FRA1 (red) of melanoma biopsy sections of patient #1. Below, nuclear FRA1 staining was quantified in three melanoma patients before and early-on therapy. f, Bioluminescent signal of A375^R-TGL cells 6 days after subcutaneous co-implantation with A375 cells expressing control or two independent short hairpins for FRA1 (n = 16 tumours) g, Seeding of A375^R-TGL cells to unlabelled tumours expressing control or two independent short hairpins for FRA1, determined by BLI (vehicle, n = 10; shFRA1#1, n = 10; shFRA1#2, n = 8 tumours). Data are presented as average; error bars represent s.e.m. P values shown were calculated using Student’s t-test (* p<0.05, ** p<0.01, **** p<0.0001).

Figure 4. The therapy-induced secretome in melanoma promotes relapse by activating the AKT pathway in resistant cells

a, Schematic diagram showing the isolation of polysome-associated transcripts from resistant cells by translating ribosome affinity profiling (TRAP) from tumours during treatment. b, Ingenuity upstream regulator analysis of gene expression profiles from A375^R cells responding to a regressing tumour microenvironment (5 days of treatment; n = 3 tumours) c, Phosphorylation status of AKT^S473 in A375^R cells, stimulated for 15 min with various CM, as indicated by immunoblotting. d, Phosphorylation status of AKT^S473 in A375^R cells after stimulation with positive regulators of the AKT pathway, up-regulated in the melanoma-TIS; ANGPTL7 (5ug/ml, 30 min; up-regulated in A375, Colo800, UACC62), PDGFD (10ng/ml, 10 min; up-regulated in Colo800), EGF (10ng/ml, 10 min; up-regulated in A375), and IGF1 (10ng/ml, 10 min; up-regulated in UACC62). e, Mice bearing A375/A375^R-TGL tumours were treated with drugs, growth of A375^R cells was followed by BLI (vehicle, n = 14; vemurafenib, n = 16; vemurafenib+BEZ235, n = 16; vemurafenib+MK2206, n = 8 tumours). f, Graphical summary of the findings. Data are presented as average; error bars represent s.e.m. P values shown were calculated using a two-tailed Mann-Whitney test.