Inhibition of MAPK p38α overcomes the cancer immunosurveillance defect caused by FPR1 loss-of-function mutation

TL;DR

A common FPR1 mutation weakens immune defenses against cancer, but this can be reversed using p38α inhibitors, improving cancer treatment outcomes.

Contribution

Identifying MAPK p38α inhibition as a therapeutic strategy to overcome FPR1 mutation-induced immunosurveillance defects.

Findings

FPR1 mutation impairs dendritic cell migration and weakens cancer immunosurveillance.

p38α inhibitors restore function of FPR1-mutant dendritic cells in vitro and in vivo.

Pharmacological p38α inhibition reduces accelerated colorectal cancer in affected mice.

Abstract

A loss-of-function polymorphism affecting the N-terminus of human formyl peptide receptor 1 (FPR1) leads to a single amino acid exchange that compromises dendritic cell (DC) migration, weakens immunosurveillance, and triggers the precocious manifestation of epithelial cancers. We present a mouse model bearing a human-mimetic mutation in FPR1 that causes the same DC defect as that observed in Fpr1 knockout animals. Genetic and pharmacological screening performed on type 1 conventional DCs (cDC1) expressing mutated FPR1 leads to the discovery that inhibitors of mitogen-activated protein kinase (MAPK) p38α correct this FPR1 defect. Small-molecule MAPK p38α inhibitors are able to restore the function of FPR1 knockout or mutated cDC1 in vitro and in vivo, hence correcting defective responses to anticancer chemotherapy or immune checkpoint blockade in mouse models. Pharmacological MAPK p38α inhibition also normalizes accelerated colorectal carcinogenesis in mice bearing an immune system affected by the absence or mutation of FPR1.

•A common FPR1 polymorphism impairs dendritic cell migration in mice and humans•MAPK p38α inhibition corrects the immune defect of FPR1-mutant dendritic cells•Targeting p38α boosts chemo- and immunotherapy efficacy in FPR1-mutant settings•Pharmacological p38α inhibition normalizes accelerated colorectal carcinogenesis

A common FPR1 polymorphism impairs dendritic cell migration in mice and humans

MAPK p38α inhibition corrects the immune defect of FPR1-mutant dendritic cells

Targeting p38α boosts chemo- and immunotherapy efficacy in FPR1-mutant settings

Pharmacological p38α inhibition normalizes accelerated colorectal carcinogenesis

An FPR1 mutation accelerates cancer onset by crippling dendritic cells and immunosurveillance. Pan et al. demonstrate that MAPK p38α inhibitors reverse this defect, restoring anti-tumor immunity and therapy responses in mice. This suggests a preventive strategy for individuals with this high-frequency immune polymorphism.

Figures

Genetic and pharmaceutic screenings to identify gene targets for restoring the *Fpr1*-deficient dendritic cell function(A and B) Schematic of the screening workflow using *Fpr1-WT* or *Fpr1*^−/−^ de-iniDCs or BMDCs for *in vitro* antigen presentation. Briefly, DCs were exposed to OVA antigen, washed, and co-cultured with B3Z T cell hybridomas; IL-2 production by B3Z cells was quantified by ELISA. The Cas9-expressing iniDCs were transfected with individual gRNAs to establish specific gene knockouts.(C–E) (C) Scatterplot of normalized IL-2 production (mean, *n* = *3*) after individual gene knock

MAPK p38α inhibition reactivates *Fpr1*-deficient dendritic cell function *in vivo*(A) Schematic of therapeutic DC vaccination. Orthotopic lung cancers were established by intravenous (i.v.) injection of luciferase-expressing TC1 (TC1-Luc) cells. The inducible immortalized dendritic cell (iniDC) precursors, either wild type (*WT*) or *Fpr1* knockout (*Fpr1*^−/−^), were differentiated into de-iniDCs, pre-pulsed with TC1 lysate, and infused intravenously. *Fpr1*^−/−^ de-iniDCs were optionally pretreated with SB203580 or skepinone-L.(B) Representative bioluminescence images of lung tumors.(C and

MAPK p38α inhibitors correct the antigen presentation defect caused by a loss-of-function *Fpr1* mutation(A) Schematic of the *in vitro* antigen presentation assay. Inducible immortalized dendritic cell (iniDC) precursors were transduced with lentivirus encoding mouse FPR1 wild type (*WT*), FPR1^T360E^ (mimicking human A346E), or empty vector and differentiated into de-iniDCs. The infected de-iniDCs were pulsed with OVA and co-cultured with B3Z T cell hybridomas. IL-2 was measured by ELISA.(B) Sequence alignment of the C-terminal region of human and mouse FPR1, highlighting the homologous muta

Transcriptomic profiling of *Fpr1*-mutant dendritic cells and their response to MAPK p38α inhibitionThe inducible immortalized dendritic cell (iniDC) precursors of the indicated *Fpr1* genotypes were differentiated, either or not treated with SB203580 (SB, 10 μM) for 24 h, and subjected to total RNA extraction for bulk RNA-seq analysis. Gene count comparisons between different *Fpr1* genotypes (A and B) or between SB203580 treatment and controls within the same genotype (C and D) were performed by k-means analysis. The top 2,000 most variable genes were used for gene clustering (A and C). Enri

Regulation of MAPK signaling pathways in dendritic cells following the treatment with MAPK p38α inhibitor(A) The inducible immortalized dendritic cells (iniDCs) of the indicated *Fpr1* genotypes were differentiated, pretreated with an ERK-inhibiting peptide (5 μM, 6 h), and then co-incubated overnight with SB203580 (10 or 30 μM). OVA-pulsed DCs were co-cultured with B3Z T cells. IL-2 production (mean ± SEM, *n* = 6) is shown as fold change relative to *Fpr1*-WT controls. Statistical significance was calculated by two-way ANOVA test.(B–E) Bone-marrow-derived dendritic cells (BMDCs) from *Fpr1-W

MAPK p38α inhibition restores the migration deficit of FPR1-mutant dendritic cells *in vitro* and *in vivo*(A–C) (A) Schematic of the *in vitro* migration assay. OVA-expressing MCA205 fibrosarcoma spheroids (stained blue, pre-treated with oxaliplatin) were overlaid with an OP9 monolayer. The differentiated inducible immortalized dendritic cells (de-iniDCs) with indicated *Fpr1* genotypes, stained with CellTracker dyes (DeepRed for untreated WT, Red for others), were pretreated with SB203580 (SB) and loaded on top of the OP9 monolayer. Cell movements were tracked via live imaging, and the acqui

Restoration of cancer immunosurveillance by MAPK p38α inhibitors in *Fpr1-*deficient animals(A) Mice bearing the *Fpr1* wild-type (*WT*) or mutant (*Fpr1*^*T360E*^) hematopoietic system were generated via bone marrow reconstitution. Subcutaneous MC38 colon cancers were established and were first intraperitoneally (i.p.) injected with SB203580 (SB) or solvent (Sol) and then received i.p. treatment with oxaliplatin (OXA), followed by PD1 blocking monoclonal antibody (aPD1) or the corresponding isotype control antibody (A).(B) Tumor sizes are reported as tumor growth curves (mean ± SEM, *n* = 6–7

Full text

Introduction

Cancer results from cumulative genetic and epigenetic alterations affecting the neoplastic cells, as well as from a permissive microenvironment reflecting the progressive failure of immunosurveillance.1 Early-onset cancers can arise in the context of iatrogenic immunosuppression required for organ transplantation, infection by human immunodeficiency virus, and inborn errors of immunity.2^,^3^,^4^,^5 Together with the clinical benefit conferred by immune checkpoint inhibitors, these observations have spurred interest in cancer immunosurveillance, which is mostly mediated by tumor-antigen-specific T lymphocytes that must be primed by dendritic cells belonging to a specific subset, the type 1 conventional dendritic cells (cDC1).6^,^7^,^8 Thus, the intratumoral presence of cDC1, together with T cells in tertiary lymphoid structures, constitutes a sign of active immunosurveillance and a favorable prognostic marker in patients bearing a variety of different cancers.9^,^10^,^11

Along similar lines, mild immune defects caused by polymorphisms in the gene coding for formyl peptide receptor 1 (FPR1) have been linked to the early diagnosis of cancer as well. Thus, a frequent loss-of-function allele affecting FPR1 (rs867228 with an allelic frequency of ∼20% for the loss-of-function variant causing a single amino acid substitution: A346E) is associated with early diagnosis of multiple types of carcinomas, as determined in a pan-cancer analysis and specifically corroborated for colorectal cancer and mammary carcinoma.12^,^13 Thus, as compared to control individuals bearing two functional FPR1 alleles (genotype: GG), individuals bearing the loss-of-function allele of rs867228 in heterozygosity (genotype G/T ∼32% of the population) and homozygosity (genotype TT, ∼4% of the population) manifest carcinomas of all types with an anticipation of ∼9 and ∼24 months, respectively. For some cancer types the impact of rs867228 on the age of onset is particularly strong, as this has been shown for luminal B breast cancer, which manifests 5 to 6 years earlier in individuals bearing the loss-of-function allele of rs867228 in heterozygosity compared to controls,12^,^13 as well as in colorectal cancer patients, in which individuals bearing the loss-of-function allele of rs867228 in homozygosity are 4–5 years younger at diagnosis than the control population.12 Evidence for a cause-effect relationship between rs867228 and early cancer diagnosis stems from mouse experiments in which knockout of Fpr1 accelerates the manifestation of mammary and colorectal cancers induced by chemical carcinogens.12^,^14 In addition, patients with breast cancer bearing rs867228 in heterozygosity exhibit poor responses to anthracycline-based adjuvant chemotherapy.15 Similarly, patients with colorectal cancer bearing rs867228 in homozygosity show poor responses to oxaliplatin-based adjuvant chemotherapy or preoperative concurrent chemoradiotherapy.15^,^16^,^17^,^18 Accordingly, in Fpr1^−/−^ mice, anticancer chemotherapy with immunogenic cytotoxicants (such as doxorubicin, oxaliplatin, or mitoxantrone) fails in conditions in which tumors implanted in wild-type (WT) controls do respond.12^,^15 Altogether, these observations, argue in favor of the clinical importance of rs867228.

Driven by these considerations, we determined the mechanisms through which FPR1 contributes to anticancer immunity. It appears that FPR1 present on the surface of cDC1 is activated in response to annexin A1 (ANXA1), a protein that is released from stressed and dying cancer cells and then determines the migration of cDC1 toward malignant cells.12^,^15 Since ANXA1 and FPR1 must act on cDC1 cells to favor cancer immunosurveillance, we designed a screening system that involves cDC1 cells derived from immortal precursors.12^,^19^,^20 Using this system, we could show that, as compared to WT cDC1 controls, Fpr1^−/−^ cDC1 cells exhibit a defect in antigen presentation that can be overcome by stimulation of Toll-like receptor 3 (TLR3) with appropriate ligands including polyinosinic:polycytidylic acid (poly I:C), polyadenylic:polyuridylic acid, and TL-532.12^,^21 Although poly I:C acid stabilized with polylysine (poly-ICLC) has successfully been employed in randomized clinical trials as an adjuvant to anticancer vaccines,22^,^23 it induces side effects including injection site reactions and systemic flu-like symptoms.24^,^25

For this reason, we decided to perform additional screens for the identification of non-toxic drugs that may correct the immune defect caused by FPR1 mutation. Here, we report the identification of mitogen-activated protein kinase (MAPK) p38α as a target, the inhibition of which corrects the FPR1 defect. This applies both to cDC1 cells or mice subjected to the Fpr1 knockout, as well as to cDC1 cells or mice bearing a point mutation in mouse Fpr1 that resembles the amino acid exchange caused by human rs867228. Pharmacological inhibitors of MAPK p38α restored natural as well chemo- or immunotherapy-induced anticancer immunosurveillance in Fpr1-deficient mice.

Results

Genetic and pharmacological screens on FPR1-deficient de-induced inducible DCs identify MAPK p38α as a target for reversing their deficient function

We previously identified 43 genes, the knockout of which improves antigen presentation by WT de-induced inducible DCs (de-iniDCs).20 Such de-iniDCs can be generated by the de-induction (or “de-immortalization” of induced immortalized DCs [iniDCs] through the removal of doxycycline and dexamethasone from the culture medium).19 IniDCs were equipped with the CRISPR Cas9 system, de-induced into de-iniDC, then exposed to the model antigen ovalbumin (OVA) and finally tested for their capacity to present the major histocompatibility complex (MHC) class I (H2K^b^)-restricted OVA-derived peptide SIINFEKL to B3Z T cell hybridoma cells. Such cells express a transgenic T cell receptor (TCR) specific for the H2K^b^-SIINFEKL complex and secrete interleukin-2 (IL-2) upon cognate stimulation,26 meaning that the concentration of IL-2 in the culture supernatant reflects the efficacy of antigen presentation by de-iniDCs.19 This system is amenable to screens for the identification of genes, the knockout of which enhances DC function (Figure 1A), as well as to pharmacological/chemical screens (Figure 1B).Figure 1. Genetic and pharmaceutic screenings to identify gene targets for restoring the Fpr1-deficient dendritic cell function(A and B) Schematic of the screening workflow using Fpr1-WT or Fpr1^−/−^ de-iniDCs or BMDCs for in vitro antigen presentation. Briefly, DCs were exposed to OVA antigen, washed, and co-cultured with B3Z T cell hybridomas; IL-2 production by B3Z cells was quantified by ELISA. The Cas9-expressing iniDCs were transfected with individual gRNAs to establish specific gene knockouts.(C–E) (C) Scatterplot of normalized IL-2 production (mean, n = 3) after individual gene knockout. Gene KOs significantly increasing IL-2 in WT, Fpr1^−/−^, or both genotypes are labeled in green, blue, and red, respectively (multiple t test). L2 production (mean ± SEM, n = 4) after treatment of de-iniDCs (D) or BMDCs (E) with MAPK p38α inhibitors SB203580 or skepinone-L (10 or 30 μM).(F) Fpr1^−/−^ de-iniDCs were pretreated with ICCB library compounds and then subjected to antigen presentation assay. The log_2_ fold change (FC) in IL-2 (n = 3) and corresponding p values (by multiple t test) are shown as volcano plot. Red dots indicate hits (FC > 2, p < 0.01).(G) Heatmap of normalized IL-2 (mean, n = 5) for selected compounds at indicated concentrations in WT and Fpr1^−/−^ de-iniDCs.(H and I) Normalized IL-2 production (mean ± SEM, n = 4) for additional Fpr1-WT and Fpr1^−/−^ de-iniDC clones treated with SB203580 or SB239063 (10 or 30 μM). Statistical significance was determined by two-way ANOVA with Dunnett’s multiple comparisons test vs. genotype-matched controls.

We transfected all guidance RNAs that improve the function of WT de-iniDCs20 into Fpr1^−/−^ iniDCs, knowing that Fpr1^−/−^ de-iniDCs only exhibit ∼30% to 50% of the antigen-presenting capacity of their WT counterparts12 (Figure 1A). Of note, knockout of three genes, Gsdmc (which codes for gasdermin C), Mapk14 (which codes for MAPK 14, also called MAPK p38α, a cytosolic Ser/Thr kinase; Figure S1A), and Ddr1 (which codes for discoidin domain receptor 1, DDR1, a receptor tyrosine kinase; Figure S1B), strongly enhanced the antigen-presenting capacity of Fpr1^−/−^ iniDCs. In contrast, other knockouts, as exemplified by Bcl2 (which codes for the venetoclax target), were unable to elevate the function of Fpr1^−/−^ iniDCs above the level of untreated WT controls (Figure 1C). In a subsequent step, we validated the knockout results by using pharmacological inhibitors of the two protein kinases. Inhibition of MAPK p38α with either SB203580 or skepinone-L improved the function of both WT and Fpr1^−/−^ de-iniDCs (Figure 1D) and bone-marrow-derived dendritic cells (BMDCs) (Figure 1E). Similarly, two different small molecule inhibitors of DDR1 enhanced the function of both WT and Fpr1^−/−^ de-iniDCs (Figure S1E).

We also performed a pharmacological screen to identify drugs that improve DC antigen-presenting function (Figure 1B). Among 480 compounds from the ICCB library, we only found three that restored antigen presentation by Fpr1^−/−^ de-iniDCs to the levels of WT DCs, when used at a concentration of 3 μM. This applies to the cell-permeable insulin receptor tyrosine kinase inhibitor hydroxy-2-naphthalenylmethylphosphonic acid tris(acetoxymethyl) ester (HNMPA-(AM)3), a plant alkaloid inhibitor of several β-glucosidases and α-glucosidases, castanospermine, and bumetanide, the latter being the most potent one (Figures 1F and 1G). Of note, the primary drug target of bumetanide, a clinically approved diuretic, is NKCC1 (also known as solute carrier family 12 member 2, SLC12A2).27 However, knockout of Nkcc1 (Figure S1C) did not improve the function of de-iniDCs, and such Nkcc1^−/−^ de-iniDCs responded to bumetanide as efficiently as WT and Fpr1^−/−^ de-iniDCs (Figure S1D), excluding the possibility that bumetanide would act on NKCC1/SLC12A2 to improve DC function. Indeed, bumetanide has been reported to mediate off-target effects including the inhibition of MAPK p38α.28^,^29^,^30^,^31 Encouraged by this information, we confirmed that classic MAPK p38α inhibitors, i.e., SB203580 and SB239063, improve antigen presentation by multiple distinct WT and Fpr1^−/−^ de-iniDC clones (Figures 1H and 1I).

In sum, convergent results from genetic and pharmacological screen indicate that inhibition of MAPK p38α can restore antigen presentation by Fpr1^−/−^ DCs in vitro. The choice to focus on MAPK p38α inhibition was also driven by the consideration that small-molecule inhibitors of MAPK p38α have been characterized in clinical trials without major signs of toxicity.32^,^33^,^34^,^35^,^36^,^37^,^38

Improved presentation of tumor antigen by SB203580-pretreated Fpr1−/− DCs in vivo

To investigate whether MAPK p38α inhibition enhances the cross presentation of tumor antigens in vivo, we submitted C57Bl/6 mice bearing orthotopic TC1 non-small cell lung cancers (NSCLC) equipped with luciferase (to allow for bioluminescence-based in vivo imaging of tumor progression) to repeated therapeutic vaccinations with de-iniDCs that were pulsed with TC1 cell lysates and then intravenously (i.v.) injected. Such de-iniDCs were either WT or Fpr1^−/−^ and were optionally pre-treated with the MAPK p38α inhibitors before they were pulsed with TC1 cell lysates (Figure 2A). As expected,12^,^15 Fpr1^−/−^ de-iniDCs were unable to induce an anticancer immune response that would lead to TC1 cancer control as the one exhibited by WT de-iniDCs. However, in vitro pretreatment of Fpr1^−/−^ de-iniDCs with SB203580 or skepinone-L before pulsing with TC1 lysates apparently improved their antigen-presenting function in vivo, allowing for tumor control (Figures 2B and 2C), and thus prolonged survival of the mice (Figure 2D). More importantly, knockout of the MAPK p38α coding gene Mapk14 improved the tumor-regressing efficacy of Fpr1^−/−^ but not WT de-iniDCs in vivo (Figures 2E–2G).Figure 2MAPK p38α inhibition reactivates Fpr1-deficient dendritic cell function in vivo(A) Schematic of therapeutic DC vaccination. Orthotopic lung cancers were established by intravenous (i.v.) injection of luciferase-expressing TC1 (TC1-Luc) cells. The inducible immortalized dendritic cell (iniDC) precursors, either wild type (WT) or Fpr1 knockout (Fpr1^−/−^), were differentiated into de-iniDCs, pre-pulsed with TC1 lysate, and infused intravenously. Fpr1^−/−^ de-iniDCs were optionally pretreated with SB203580 or skepinone-L.(B) Representative bioluminescence images of lung tumors.(C and D) Tumor growth curves (mean ± SEM, photon flux) and Kaplan-Meier survival plots for mice receiving the indicated DC vaccines (n = 8–10 mice/group). Statistical significance was calculated by type II ANOVA (C) and log rank test (D).(E) Schematic for genetic rescue. Mapk14 was knocked out via transfecting gRNAs targeting Mapk14 (sgRNA-Mapk14, mixture of two sequences) in Cas9-expressing iniDCs. A non-targeting gRNA (sgRNA-NT) was used as assay control. The resulting de-iniDCs were pulsed with TC1 lysate and infused into tumor-bearing mice.(F and G) Tumor growth (mean ± SEM) and survival of mice receiving DCs with the indicated genotypes and sgRNA transfections (n = 10–12 mice/group). Statistical analysis was calculated by type II ANOVA (F) and log rank test (G).

We therefore investigated the impact of WT, untreated, and SB203580-treated Fpr1^−/−^ de-iniDCs on the abundance and activation state of T lymphocyte subsets by means of high-dimensional immunofluorescence cytometry (Figure S2A). As compared to WT controls, the lungs of mice that received untreated Fpr1^−/−^ de-iniDCs exhibited a reduction of T cells with a central memory phenotype (T_CM_, CD44^hi^CD62L^hi^) among CD4^+^ T cells, as well that of T cells with an effector memory phenotype (T_EM_, CD44^hi^CD62L^lo^) both among CD4^+^ and CD8^+^ T cells. This defect was completely reversed by SB203580 treatment of Fpr1^−/−^ de-iniDCs (Figures S2B and S2D). Injection of SB203580-pretreated Fpr1^−/−^ de-iniDCs generally had positive effects on the abundance of T_CM_ and T_EM_ in the spleen and peripheral blood, enhanced expression of activation/exhaustion markers (such as CD69, CTLA-4, ICOS and PD-1) (Figures S2B and S2D), and also enhanced the percentage of CD4^+^ and CD8^+^ T cells expressing granzyme B, various cytokines (interferon gamma [IFN-γ], interleukin-2 [IL-2], IL-17, tumor necrosis factor alpha [TNF-α]), and the proliferation marker Ki67 (Figures S2C and S2E).

Altogether, these results confirm the idea that pretreatment of Fpr1^−/−^ de-iniDCs with MAPK p38α inhibitors improves immunostimulatory functions and reverses the immune defect caused by Fpr1 knockout.

Tumors implanted in Fpr1^−/−^ mice fail to respond to immunogenic chemotherapy with the combination of cyclophosphamide (CTX) and mitoxantrone (MTX), although this treatment slowed tumor progression in WT mice.12 Treatment of orthotopic MCA205 fibrosarcomas by injection of the prototypic MAPK p38α inhibitor SB203580 had little effect on its own, but fully restored tumor growth inhibition in Fpr1^−/−^ mice (Figures S2F and S2G), supporting the idea that MAPK p38α inhibition can correct the immunodeficiency caused by defective FPR1 signaling.

Defective antigen presentation by Fpr1T360E DCs is reversed by MAPK p38α inhibition in vitro and in vivo

The experiments mentioned above were performed in Fpr1^−/−^ de-iniDCs. We attempted to build a model that more accurately reflects the human loss-of-function polymorphism rs867228 of FPR1 by substituting the endogenous mouse Fpr1 gene by a point-mutated Fpr1 gene (Figure S3A). The more frequent (functional) allele of human FPR1 bears an alanine (A) residue in position 346, while the less frequent (dysfunctional) allele carries a substitution with a glutamic acid (E) residue in this position.15 Alignment of the human and mouse N-termini led to the conclusion that the mutation that would most closely mimic the substitution in human FPR1 would be a threonine (T) to glutamic acid (E) replacement in mouse Fpr1 in position 360 (T360E) (Figures 3A and 3B). Molecular modeling suggested that the human FPR1 substitution and the mouse Fpr1 T360E mutation do not affect the overall structure of this G-protein-coupled seven-transmembrane receptor and only impinge marginally on the (relatively unstructured) C-terminus of the molecule in the cytosol (Figure S3A). Of note, transduction of Fpr1^−/−^ DCs with WT FPR1-expressing lentivirus restored their function more efficiently than did FPR1^T360E^ (Figures 3A–3C), suggesting that the FPR1 T360E mutation indeed confers a loss-of-function phenotype.Figure 3MAPK p38α inhibitors correct the antigen presentation defect caused by a loss-of-function Fpr1 mutation(A) Schematic of the in vitro antigen presentation assay. Inducible immortalized dendritic cell (iniDC) precursors were transduced with lentivirus encoding mouse FPR1 wild type (WT), FPR1^T360E^ (mimicking human A346E), or empty vector and differentiated into de-iniDCs. The infected de-iniDCs were pulsed with OVA and co-cultured with B3Z T cell hybridomas. IL-2 was measured by ELISA.(B) Sequence alignment of the C-terminal region of human and mouse FPR1, highlighting the homologous mutations (human A346E and mouse T360E).(C) IL-2 production (mean ± SEM, n = 5) from the assay in (A). Statistical significance was calculated by two-way ANOVA with Dunnett’s test.(D) Schematic of therapeutic DC vaccination. Orthotopic lung cancers were established by intravenous (i.v.) injection of luciferase-expressing TC1 (TC1-Luc) cells. Fpr1-WT or Fpr1^T360E^ iniDCs were differentiated into de-iniDCs, pulsed with TC1 lysate with or without SB203580 pretreatment, and infused intravenously.(E and F) Tumor growth curves (mean ± SEM, photon flux) and Kaplan-Meier survival plots for mice receiving the indicated DC vaccines (n = 10–12 mice/group). Statistical significance was calculated by type II ANOVA test (E) or log rank test (F). De-iniDCs of the indicated Fpr1 genotypes were treated with SB203580 (SB, 10 μM) or skepinone-L (SKL, 10 μM) for 4 or 8 h before lysis.(G) Representative western blots for phosphorylated MAPK p38α (p-p38α), total p38α, phosphorylated MK2 (p-MK2), total MK2, and β-actin (loading control).(H–K) Quantified ratios (mean ± SEM, n = 3 independent experiments) of p-p38α (H), total p38α (I), p-MK2 (J), and total MK2 (K) to β-actin (K). Statistical analysis was calculated by one-way ANOVA test.

We therefore generated iniDCs bearing the Fpr1^T360E^ knockin mutation in homozygosity (genotype: Fpr1^T360E/T360E^) (Figure S3B) and characterized their phenotype after their conversion into de-iniDCs, which was similar to that of Fpr1^−/−^ de-iniDCs, including downregulation of multiple activation markers (CD40, CD80, CD86, CD103, and CD183), reduced expression of MHC class II molecules, decreased cytokine production (IL-1b and IL-6), reduced OVA antigen presentation in vitro (leading to IL-2 production by B3Z cells), and enhanced PD-L1 expression (Figure S3C). The MAPK p38α inhibitor SB203580 similarly reversed such defects in Fpr1^−/−^ and Fpr1^T360E/T360E^ de-iniDCs (Figure S3D).

Stimulated by these results, we generated mice bearing a germline Fpr1^T360E/T360E^ mutation and then derived BMDC from such mice to compare their phenotype to WT controls. Similar to Fpr1^T360E/T360E^ de-iniDCs, such Fpr1^T360E/T360E^ BMDCs exhibited a reduction in the expression of activation markers (CD80, CD86, CD103, and CD83) and cytokine production (IL-12 p70 and TNF-α) (Figure S3E), reduced antigen presentation (Figure S3F), as well as reversal of the phenotype by in vitro treatment with SB203580 (Figures S3F and S3G). Of note, compared with WT controls, Fpr1^T360E/T360E^ and Fpr1^−/−^ de-iniDCs exhibited reduced T cell activation when stimulated with either soluble OVA (Figure S3H) or killed OVA-expressing MCA205 murine fibrosarcoma cells (Figure S3I) but not when stimulated with the OVA SIINFEKL peptide (Figure S3J). This suggests that the Fpr1-deficient de-iniDCs have lost the capacity of presenting tumor antigens but are endowed with full direct T cell activation capacities. In addition, defective presentation of soluble OVA protein and cancer-cell-associated OVA could be corrected by MAPK p38α inhibitors (Figures S3H and S3I). As a control, MAPK p38α inhibitors did not improve B3Z T cell stimulation by iniDCs that had been rendered MHC-I-deficient due to the knockout of the gene coding for β-2 microglobulin (genotype: B2m^−/−^) (Figure S3J). Moreover, when WT de-iniDCs were exposed to pharmacological FPR1 inhibitors such as cyclosporin H and HCH6-1, they exhibited deficient antigen presentation, and this defect could be restored by MAPK p38α inhibitors (Figure S3K).

Of note, as compared to WT de-iniDCs, OVA-pulsed Fpr1^−/−^ and Fpr1^T360E/T360E^ de-iniDCs that were subcutaneously (s.c.) inoculated (Figure S4A) exhibited a similar defect in eliciting in vivo immune responses measured by quantifying the percentage of CD8 T cells carrying a H2^b^/SIINFEKL-specific T cell receptor (TCR), exhibiting a T_CM_ phenotype, producing IL-2, or expressing the proliferation marker Ki67 in the draining lymph node and the spleen (Figures S4B and S4C). In vitro pretreatment of Fpr1^−/−^ and Fpr1^T360E/T360E^ de-iniDCs before pulsing with OVA improved their immunostimulatory function in vivo (Figure S4C).

To assess OVA-specific T cell priming in vivo, we immunized WT vs. Fpr1 mutant mice (after optional treatment with MAPK p38α inhibitor) with killed MCA205-OVA cell and then collected peripheral blood cells and splenocytes to stimulate them in vitro with the MHC-I-restricted OVA peptide SIINFEKL, followed by the detection of SIINFEKL-reactive T cells by means of an IFN-γ ELISPOT assay (Figure S5A). Blood cells and splenocytes from Fpr1 mutant mice displayed markedly reduced numbers of IFN-γ-producing spots compared to WT controls, indicating impaired antigen presentation and T cell activation in vivo (Figures S5B and S5C). Notably, systemic treatment of Fpr1 mutant mice with SB203580 restored ELISPOT responses (Figures S5B and S5C). We also tested SB203580 in a classical in vivo antigen presentation assay (Figures S5D and S5E). We found that adoptively transferred OT1 T cells are less efficiently primed in Fpr1 mutant mice, as compared to WT mice, following vaccination with killed MCA205-OVA cells (Figures S5F and S5H). Accordingly, as compared to WT controls, Fpr1 mutant de-iniDCs pulsed with TC1 cell lysates and then intravenously (i.v.) injected into mice bearing orthotopic TC1 lung cancers were relatively inefficient in conferring tumor control (Figures 3D–3F). Pretreatment of Fpr1^T360E^ de-iniDCs with SB203580 in vitro before pulsing them with TC1 lysates improved tumor control in vivo (Figure 3E) and thus prolonged the survival of the mice (Figure 3F).

We also tested the effect of a chemical library containing MAPK inhibitors to observe that multiple MAPK p38α inhibitors can reverse deficient antigen presentation by both Fpr1^−/−^ and Fpr1^T360E/T360E^ de-iniDCs (Figures S5I and S5J). This also applies to two positive controls, the tool compounds SB203580 and SB239063, as well as to four clinically tested MAPK p38α inhibitors, doramapimod (which reportedly mitigates chronic obstructive pulmonary disease),32 losmapimod (which improves muscle function in facioscapulohumeral muscular dystrophy),37 pamapimod (active against rheumatoid arthritis but discontinued due to toxicity),33 and ralimetinib (evaluated in cancer patients).38 Hence, various distinct MAPK p38α inhibitors indistinguishably restore the function of Fpr1^−/−^ and Fpr1^T360E/T360E^ DCs.

We conclude that a point mutation in mouse Fpr1 mimicking rs867228 affecting human FPR1 disrupts the function of DCs and that this immune defect can be reversed by MAPK p38α inhibition.

Mechanisms of the DC defect caused by Fpr1 mutation and its reversal by MAPK p38α inhibition

We observed that both MAPK p38α and its direct downstream substrate MAPK-activated protein kinase 2 (MK2) are hyperphosphorylated in Fpr1^KO^ and Fpr1^T360E^ de-iniDCs unless they were treated with either of the two MAPK p38α inhibitors, SB203580 or skepinone-L (Figures 3G–3K). In addition, we performed RNA sequencing on de-iniDCs with different genotypes (WT, Fpr1^−/−^, and Fpr1^T360E/T360E^) to gain insights into the immune defect caused by the absence or mutation of Fpr1. Gene Ontology (GO) terms of genes that were lost both in Fpr1^−/−^ and Fpr1^T360E/T360E^ de-iniDCs compared to WT DCs were mostly dealing with cellular motility including chemotaxis and migration (Figures 4A and 4B). Conversely, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that both Fpr1^−/−^ and Fpr1^T360E/T360E^ de-iniDCs commonly downregulated multiple mRNAs involved in the MAPK and TNF signaling pathways (Figures S6A–S6D). The downregulation of genes related to cell migration was also observed when BMDCs from Fpr1^−/−^ and Fpr1^T360E/T360E^ mice were compared to BMDC from WT and Fpr1^+/−^ control animals at the level of GO terms (Figures S6E and S6F). At the level of KEGG pathway analyses, genes related to cytokine-cytokine receptor interactions and IL-17 signaling were commonly downregulated in Fpr1^T360E/T360E^ BMDC compared to WT controls (Figures S6G and S6H), as well as in Fpr1^−/−^ compared to Fpr1^+/−^ controls (Figures S6I and S6J). We also identified mRNAs that are commonly upregulated in both Fpr1^−/−^ and Fpr1^T360E/T360E^ de-iniDCs treated with the MAPK p38α inhibitor SB203580. Such mRNAs were linked to cellular locomotion and adhesion according to GO term analyses (Figures 4C and 4D), as well as to G-protein-coupled receptor (GPCR) signaling, chemokine signaling, and adhesion according to KEGG analyses (Figure 4E).Figure 4. Transcriptomic profiling of Fpr1-mutant dendritic cells and their response to MAPK p38α inhibitionThe inducible immortalized dendritic cell (iniDC) precursors of the indicated Fpr1 genotypes were differentiated, either or not treated with SB203580 (SB, 10 μM) for 24 h, and subjected to total RNA extraction for bulk RNA-seq analysis. Gene count comparisons between different Fpr1 genotypes (A and B) or between SB203580 treatment and controls within the same genotype (C and D) were performed by k-means analysis. The top 2,000 most variable genes were used for gene clustering (A and C). Enriched pathways for each cluster of interest are shown in (B and D). The adjusted p values are labeled next to its corresponding circle whose size indicates the number of genes in this pathway. Genes that are commonly upregulated (E) and downregulated (F) with significantly statistical differences (FC > 2, adjusted p < 0.01) between Fpr1^T360E^ vs. Fpr1^T360E^ + SB and Fpr1^−/−^ vs. Fpr1^−/−^ + SB were subjected to KEGG pathway mapping as shown in (E and F). Gene enrichment is shown for the indicated pathways (dot size indicates the number of genes, and dot color represents the estimated statistical differences [false discovery rate, FDR]).

Inspired by these gene expression analyses, as well as the fact that FPR1 signaling has previously been linked to both the MAPK p38α and ERK pathways,39^,^40^,^41 we investigated the effects of ERK signaling on the reconstitution of antigen presentation through MAPK p38α inhibition by SB203580 of Fpr1^−/−^ and Fpr1^T360E/T360E^ de-iniDCs. Of note, when MAPK p38α inhibition was combined with an ERK inhibitor, the beneficial effect of MAPK p38α inhibition was compromised both in Fpr1^−/−^ and Fpr1^T360E/T360E^ de-iniDCs (Figure 5A). When compared to WT controls, BMDC from Fpr1^T360E/T360E^ mice failed to activate the phosphorylation of ERK1/2 in response to two FPR1 agonists, namely the formulated tripeptide N-formyl-methionine-leucyl-phenylalanine (fMLP) and recombinant annexin A1 (rANXA1) protein (Figures 5B–5E). In both cases, ERK1/2 activation was restored by MAPK p38α inhibition with SB203580 (Figures 5B–5E). Consistently, de-iniDCs bearing either Fpr1^−/−^ or Fpr1^T360E/T360E^ genotypes also exhibited reduced ERK1/2 activation upon rANXA1 stimulation, which was restored by the MAPK p38α inhibitors SB203580 or skepinone-L (Figures S7A–S7D and S7F–S7J). The improvement in antigen presentation observed in Fpr1^−/−^ and Fpr1^T360E/T360E^ DCs after SB203580 or skepinone-L treatment was also abolished by the small molecular ERK inhibitor PD98059 (Figure S7E). Altogether, these results suggest that Fpr1 mutation compromises ERK1/2 signaling, which can be restored by MAPK p38α inhibition. This signaling via ERK1/2 appears essential for DC function, echoing prior reports on the critical role of ERK signaling in DC maturation and function.42^,^43Figure 5. Regulation of MAPK signaling pathways in dendritic cells following the treatment with MAPK p38α inhibitor(A) The inducible immortalized dendritic cells (iniDCs) of the indicated Fpr1 genotypes were differentiated, pretreated with an ERK-inhibiting peptide (5 μM, 6 h), and then co-incubated overnight with SB203580 (10 or 30 μM). OVA-pulsed DCs were co-cultured with B3Z T cells. IL-2 production (mean ± SEM, n = 6) is shown as fold change relative to Fpr1-WT controls. Statistical significance was calculated by two-way ANOVA test.(B–E) Bone-marrow-derived dendritic cells (BMDCs) from Fpr1-WT or Fpr^T360E^ mice were pretreated with SB203580 (10 μM, 4 h) and stimulated with FPR1 ligands recombinant annexin A1 (rANXA1, B and D) or N-formylmethionine-leucyl-phenylalanine (fMLP, C,E) for another 4 h. (B and C) Representative western blots for phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2, and β-actin (loading control). (D and E) Quantification of the p-ERK1/2/ERK1/2 ratio (mean ± SEM, n = 3 independent experiments, with three mice per group) normalized as fold change to unstimulated Fpr1-WT controls. Statistical analysis was calculated by two-way ANOVA test.

In the next step, we investigated whether the defective migration of Fpr1-deficient de-iniDCs can be modeled in vitro. For this, we optimized a coculture system consisting of a monolayer of unlabeled fibroblast-like OP9 cells, sandwiched with mobile de-iniDCs of different genotypes (WT labeled with CellTracker Deep Red plus Fpr1^−/−^ or Fpr1^T360E/T360E^ labeled with CellTracker Red), as well as a semi-spheroid of MCA205 fibrosarcoma cells immobilized in Matrigel that were previously stained with CellTracker blue and treated with the ICD inducer oxaliplatin (Figure 6A). Fluorescence videomicroscopy allowed to track the movement of de-iniDCs that approached the dying cancer cells (Figure 6B). WT de-iniDCs cells exhibited a superior capacity to approach the cancer cells, as well as higher maximum travel velocities, compared to Fpr1^−/−^ and Fpr1^T360E/T360E^ de-iniDCs (Figures 6C and S7K; Video S1). Tumor invasion and maximum velocity of Fpr1^−/−^ and Fpr1^T360E/T360E^ de-iniDCs were increased by pretreatment with SB203580 (Figures 6C and S7K; Video S2). These DC-motility-restoring effects of SB203580 were lost when an ERK inhibitory peptide was added (Figures S8A and S8B). Similarly, the DC-motility-restoring effects of the MAPK p38α inhibitor skepinone-L were abrogated by the ERK small molecular inhibitor PD98059 (Figures S8C and S8D).Figure 6MAPK p38α inhibition restores the migration deficit of FPR1-mutant dendritic cells in vitro and in vivo(A–C) (A) Schematic of the in vitro migration assay. OVA-expressing MCA205 fibrosarcoma spheroids (stained blue, pre-treated with oxaliplatin) were overlaid with an OP9 monolayer. The differentiated inducible immortalized dendritic cells (de-iniDCs) with indicated Fpr1 genotypes, stained with CellTracker dyes (DeepRed for untreated WT, Red for others), were pretreated with SB203580 (SB) and loaded on top of the OP9 monolayer. Cell movements were tracked via live imaging, and the acquired time stacks were analyzed by ImageJ to quantify the migratory potential of the de-iniDCs. Representative tracks of migrating de-iniDCs are shown in (B). Mean velocity of tracked cells are shown in (C). Each dot represents a single DC; data pooled from three wells per condition. The quantification and comparison of mutant de-iniDCs or KO de-iniDCs were always compared with differently labeled WT de-iniDCs in the same well. Statistical significance was calculated by one-way ANOVA test.(D and E) (D) Schematic of the in vivo DC migration assay. Mice with orthotopic TC1-Luc lung cancer received i.v. injections of TC1 lysate-pulsed de-iniDCs (WT or Fpr1^−/−^). Where indicated, the Fpr1^−/−^ de-iniDCs were pretreated with SB203580 for 4 h. Three days after de-iniDC injection, the tumor-bearing lungs were subjected to immunohistochemical detection of CRISPR-Cas9-expressing cells. Representative immunohistochemistry images showing the presence of Cas9-expressing DCs in lung tumors (indicated by arrows) are provided in (E). Scale bars: 100 μm.(F) Quantification of Cas9^+^ de-iniDC presence in tumor regions via QuPath analysis (n ≥ 16 mice/group). Statistical significance was calculated by one-way ANOVA test.(G–I) Competitive migration assay. Differently CellTracker-labeled de-iniDCs (Fpr1-WT, Fpr1^−/−^, Fpr1^T360E^) were mixed and injected i.v. into tumor-bearing mice. Lungs were collected 24 h after DC injection and dissociated into single-cell suspensions for flow cytometry. Gating of different CellTracker-labeled DCs is depicted in (H), and quantification of DC abundance (ration in viable leukocytes) is shown as boxplot (I, n = 10 mice/group). Statistical analysis was calculated by one-way ANOVA test.(J–M) PBMCs from healthy donors were genotyped FPR1 Rs867228 (E346A) via TaqMan allelic discrimination assays, where VIC fluorescence indicates the reference allele (G) and FAM fluorescence indicates the variant allele (T). Monocyte-derived DCs (moDCs) from genotyped donors were treated with SB or skepinone-L (SKL) and assessed in the in vitro migration assay toward U2OS-OVA tumoroids. DC travel displacement (K), maximum distance (L), and speed (M) were quantified and their median values obtained from each PBMC sample were plotted (n = 4/5). Statistical analysis was calculated by paired t test (treatment vs. control per genotype) or unpaired t test (genotype comparison).

Video S1. Representative video showing the movement of DCs without treatment, related to Figure 6. Red color indicates CellTracker DeepRed^+^ WT DCs imaged with a Cy5 filter set; green color indicates CellTracker Red^+^Fpr1^−/−^ DCs imaged with a Cy3 filter set; and blue color indicates CellTracker Blue^+^ dying MCA205 cells imaged with a DAPI filter set.

Video S2. Representative videos showing the movement of DCs pretreated with SB203580, related to Figure 6. Red color indicates CellTracker DeepRed^+^ WT DCs imaged with a Cy5 filter set; green color indicates CellTracker Red^+^Fpr1^−/−^DCs imaged with a Cy3 filter set; and blue color indicates CellTracker Blue^+^ dying MCA205 cells imaged with a DAPI filter set.

We also determined the capacity of de-iniDCs to enter TC1 NSCLC tumor lesions after i.v. injection by immunohistochemical detection of Cas9 expressed by the de-iniDCs (Figures 6D and 6E). Of note, as compared to WT controls, Fpr1^−/−^ de-iniDCs were deficient in their capacity to attain the center of TC1 cancer nodules in the lung, and this deficiency was corrected by their in vitro pre-treatment with SB203580 (Figure 6F). To confirm this result, we labeled de-iniDCs with different Fpr1 genotypes with fluorescent CellTrackers, treated them with MAPK p38α inhibitors, and then stimulated them with TC1 cell lysates in vitro before injecting them into TC1-lung-cancer-bearing mice. Both SB203580 and skepinone-L enhanced the intratumoral accumulation of de-iniDCs, as detectable by flow cytometry (Figures 6G and 6H). The result of this experiment strengthened the conclusion that MAPK p38α inhibition enhances the intratumoral recruitment of Fpr1-deficient or -mutated DCs (Figure 6I).

Next, we characterized the mobility of human DCs bearing the FPR1 A346E mutation. For this, we obtained peripheral blood mononuclear cells (PBMCs) from healthy donors and genotyped rs867228. Among 10 donors, we identified four individuals bearing a heterozygous A346E mutation of FPR1 (Figure 6J). Such samples were used to generate monocyte-derived DCs, which then were tested in DC migration/motility assays. We found that the human FPR1 A346E heterozygous mutation yields a similar phenotype for DCs as the mouse Fpr1 T360E mutation, namely a relative migratory defect that can be improved by MAPK p38α inhibition (Figures 6J–6M andS8E–S8G). Hence, we conclude that the mouse FPR1 T360E mutation faithfully mimics the human A346E variant.

Altogether, it appears that the defective migration of de-iniDCs into cancers is ameliorated by MAPK p38α inhibition.

Defective immunosurveillance caused by Fpr1 mutation is reversed by MAPK p38α inhibition in vivo

The aforementioned results indicate that the immune defect caused by the Fpr1^T360E/T360E^ mutation, which mimics the patient-relevant substitution in human FPR1, can be corrected by MAPK p38α inhibition. To corroborate this hypothesis in vivo, we explored two models of cancer immunosurveillance in which we reconstituted the hematopoietic system of lethally irradiated WT C57BL/6 mice by transplanting hematopoietic stem cells from the bone marrow of WT, Fpr1^−/−^, or Fpr1^T360E/T360E^ mice to induce an immune-system-specific Fpr1 defect.

In the first model, mice bearing a WT or Fpr1^T360E/T360E^ immune system were s.c. injected with MC38 murine colon adenocarcinoma cells, which were later treated with vehicle alone, SB203580, oxaliplatin, PD-1 blockade, or their combinations (Figure 7A). SB203580 alone only showed tumor-growth-reducing effects in Fpr1^T360E/T360E^ mice but not in WT mice (Figure 7B). Oxaliplatin or PD-1 blockade alone, as well as their combinations, efficiently reduced tumor progression and extended animal survival in WT mice but not in Fpr1^T360E/T360E^ mice (Figure 7B). Importantly, in mice bearing an Fpr1^T360E/T360E^ mutated immune system, treatment with SB203580 significantly improved the outcome of oxaliplatin, PD-1 blockade, and their combination (Figure 7B).Figure 7. Restoration of cancer immunosurveillance by MAPK p38α inhibitors in *Fpr1-*deficient animals(A) Mice bearing the Fpr1 wild-type (WT) or mutant (Fpr1^T360E^) hematopoietic system were generated via bone marrow reconstitution. Subcutaneous MC38 colon cancers were established and were first intraperitoneally (i.p.) injected with SB203580 (SB) or solvent (Sol) and then received i.p. treatment with oxaliplatin (OXA), followed by PD1 blocking monoclonal antibody (aPD1) or the corresponding isotype control antibody (A).(B) Tumor sizes are reported as tumor growth curves (mean ± SEM, n = 6–7 mice/group). Statistical significance was calculated by two-way ANOVA test.(C–E) Mice bearing Fpr1-WT, Fpr1^−/−^, or Fpr1^T360E^ hematopoietic systems were subjected to colorectal carcinogenesis with azoxymethane (AOM)/dextran sulfate sodium (DSS) (C). During the induction phase, SB or Sol was administrated weekly. Ilea were subjected to H&E staining, and the tumor region was detected and quantified with QuPath. (D) Representative H&E-stained images of ileum Swiss rolls showing tumor lesions. Scale bars: 1 mm. (E) Quantification of total tumor lesions per mouse (n ≥ 10 mice/group). Statistical significance was calculated by one-way ANOVA test.

In the second model, mice bearing a WT, Fpr1^−/−^, or Fpr1^T360E/T360E^ immune system were treated with two carcinogens, azoxymethane (AOM, which was injected once i.p.) and dextran sulfate sodium (DSS, which was administered orally), that together cause inflammation-associated colorectal cancer44 (Figure 7C). As previously reported for mice bearing a germline Fpr1^−/−^ defect,14 mice with an Fpr1^−/−^ or Fpr1^T360E/T360E^ immune system were particularly prone to AOM/DSS colorectal carcinogenesis, meaning that they exhibited a higher number of adenomatous lesions than WT controls (Figure 7D). Weekly injections of SB203580 had no effects on the number of tumoral lesions per mouse in WT controls but significantly reduced tumorigenesis in mice transplanted with Fpr1^−/−^ or Fpr1^T360E/T360E^ hematopoietic stem cells (Figure 7E).

In conclusion, it appears that the prototypic MAPK p38α inhibitor SB203580 can correct the defect in immunosurveillance that affects Fpr1-deficient mice.

Discussion

Homozygosity in the minor allele of rs867228 (FPR1^A346E/A346E^, 4% of the population) is associated with an acceleration of carcinoma diagnosis by 2 years and heterozygosity (FPR1^A346E^, 32% of the population) by 9 months, compared to the majority of the population bearing the two “normal” alleles.12 This loss translates to ∼600 million carcinoma-free years for the world’s population.45 In addition, rs867228 appears to have a negative impact on treatment responses in some cancers.15^,^16^,^17^,^18 These considerations justify efforts to correct the defect associated with rs867228 affecting FPR1.

FPR1, a myeloid-expressed pattern recognition receptor, responds to bacterial formylated peptides and endogenous ligands like mitochondrial peptides and ANXA1.46^,^47 Consequently, Fpr1^−/−^ mice show increased susceptibility to infections by Escherichia coli40 or Listeria monocytogenes48 and impaired bone fracture healing.49 Conversely, Fpr1^−/−^ mice are resistant to Yersinia pestis infection, which uses FPR1 as a receptor,50 and to various inflammatory conditions including endotoxin-induced lung injury,51 bleomycin-induced pulmonary fibrosis,52 smoke-induced emphysema,53 ischemic or oxygen-induced retinopathy,54 and transient focal brain ischemia.55 These context-dependent detrimental effects, particularly on infection susceptibility, may underlie the prevalence of FPR1 loss-of-function polymorphisms.47^,^50 During sterile injury, FPR1 stimulation by leaked mitochondrial peptides and ANXA1 exacerbates neutrophil infiltration,47^,^51^,^52^,^54^,^55 which is associated to enhanced MAPK p38α activation.56^,^57^,^58 Accordingly, this FPR1-triggered activation of both p38α and ERK coordinates neutrophil chemotaxis, with ERK providing a “go” and p38α a “stop” signal for accurate target approach.39

In cancer, FPR1 acts on DCs to facilitate their migration toward dying cancer cells and the cross-presentation of tumor antigens to T cells.12^,^15 Here, we used complementary genetic and pharmacological screens to identify strategies restoring the function of Fpr1^−/−^ DCs. We screened a limited number of knockouts that reportedly improve the function of WT DCs,12 finding that knockout of Mapk14 (which encodes MAPK p38α) improved antigen presentation by Fpr1^−/−^ DCs. Multiple MAPK p38α inhibitors also restored antigen presentation by Fpr1^−/−^ DCs, including experimental/clinical inhibitors like doramapimod, losmapimod, pamapimod, ralimetinib, SB203580, and skepinone-L, as well as the diuretic bumetanide (via an off-target p38α effect). Using the well-characterized inhibitors SB203580 and skepinone-L, we demonstrated that pharmacological p38α inhibition corrects deficient cancer immunity promotion by Fpr1^−/−^ DCs in vivo, including therapeutic DC vaccination and PD-1 blockade against NSCLC and fibrosarcomas.

Here, we developed a mouse model harboring the Fpr1^T360E^ mutation, which mimics the human rs867228 polymorphism. This mutation alters the receptor’s cytosolic N-terminus, likely sparing ligand binding but disrupting function by promoting monomerization and constitutive activation, rendering it ligand-refractory.59^,^60^,^61 Accordingly, Fpr1^T360E^ DCs failed to activate ERK1/2 in response to agonist FPR1 ligands including inflammation-relevant formylated peptide and cancer-relevant ANXA1. Notably, MAPK p38α inhibition restored ERK1/2 activation in these cells. Thus, suppressing the p38α “stop” signal can reinstate the ERK1/2 “go’ signal39 in the context of this loss-of-function FPR1 mutation.

Inhibition of MAPK p38α restored the deficient migration of Fpr1^−/−^ or Fpr1^T360E^ DCs in the presence of dying cancer cells in vitro, in a coculture system under videofluorescence microscopic observation. Similar observations were obtained for human-monocyte-derived DCs bearing the FPR1^A346E^ genotype. Wild-type DC migration was unaffected. Pre-treating mouse with Fpr1-deficient DCs with p38α inhibitors enhanced their intratumoral accumulation in vivo. Concomitantly, MAPK p38α inhibition restored deficient immunotherapy effects of PD-1 blockade against non-small cell lung cancer (NSCLC) in mice with Fpr1^−/−^ or Fpr1^T360E^ immune systems. Moreover, MAPK p38α inhibition restored natural immunosurveillance in mice with Fpr1^−/−^ or Fpr1^T360E^ immune systems. Thus, Fpr1^−/−^ or Fpr1^T360E^ mice developed more neoplastic lesions in a model of inflammation-induced colic carcinogenesis than WT mice, and this acceleration of colic oncogenesis was abolished by MAPK p38α inhibition. Of note, the mechanism of p38α inhibitors appears distinct from TLR3 ligands. Poly I:C reportedly activates both MAPK p38α and ERK1/2 in human microglia and multiple myeloma cells,62^,^63 but neither MAPK p38α nor ERK1/2 in DCs or macrophages.64

It is worthwhile noting that modern pharmacological MAPK p38α inhibitors have little side effects, although their initial use as anti-inflammatory drugs against various diseases including rheumatoid arthritis,34 arterial inflammation,35 and chronic obstructive pulmonary disease36 turned out to be deceptive. Similarly, bumetanide has little side effects, prompting its (unsuccessful) use against autism spectrum disorders in children and adolescents,65 as well as against Parkinson disease in adults.66 This supports their potential repurposing for cancer prevention or therapy in individuals with the loss-of-function FPR1 polymorphism. While p38α inhibitors have failed in prior oncology trials such as for the treatment of advanced or metastatic cancers67 or ovarian cancer,38 none have been tested for enhancing immune checkpoint blockade. Future trials evaluating this combination should consider stratifying patients by FPR1 genotype.

In conclusion, we identified a promising pharmacological strategy, consisting in the inhibition of MAPK p38α, to correct the immune defect caused by a frequent loss-of-function polymorphism of FPR1 that weakens cancer immunosurveillance, accelerates the manifestation of carcinomas, and compromises the efficacy of antineoplastic treatments.

Limitations of the study

Our work provides evidence that the human FPR1^A346E^ defect is phenocopied by the mouse Fpr1 ^T360E^ mutation, causing a DC migration defect reversible by MAPK p38α inhibition in vitro. Beyond this similarity, however, our work is fully mouse-centric, revealing defective DC functions with regard to cancer immunosurveillance that are similar in their phenotype for mice bearing a full Fpr1 knockout and the Fpr1 T360E mutation. In such mice, MAPK p38α inhibition in DCs restored cancer immunosurveillance and chemoimmunotherapy responses to those observed in WT animals. Nonetheless, these results do not demonstrate that such a strategy would work in patients affected by the frequent rs867228 polymorphism in FPR1. Clinical trials stratifying patients by FPR1 genotype are required to test p38α inhibitors in this population. The feasibility and cost-effectiveness of such a precision immunotherapy strategy remain to be determined.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Peng Liu (peng.liu@inserm.fr).

Materials availability

This study did not generate new unique reagents.

Data and code availability

• •RNA-seq data have been deposited in the European Nucleotide Archive (ENA) and are publicly available under the accession number PRJEB102824 (https://www.ebi.ac.uk/ena/browser/view/PRJEB102824).
• •This paper does not report original code.
• •Any additional information required to reanalyze the data reported in this work paper is available from the lead contact upon request.

Acknowledgments

The primary sponsor of this project is the Cancer Research ASPIRE Award from the 10.13039/100014599Mark Foundation. G.K. is supported by the 10.13039/501100004099Ligue Contre le Cancer (équipe labellisée); Agence National de la Recherche (ANR-22-CE14-0066 VIVORUSH, ANR-23-CE44-0030 COPPERMAC, ANR-23-R4HC-0006 Ener-LIGHT); 10.13039/100007391Association pour la Recherche sur le Cancer (ARC); 10.13039/501100006431Cancéropôle Île-de-France; 10.13039/501100002915Fondation pour la Recherche Médicale (FRM); a donation by Elior; European Joint Programme on Rare Diseases (EJPRD) Wilsonmed; European Research Council Advanced Investigator Award (ERC-2021-ADG, grant no. 101052444; project acronym: ICD-Cancer); The ERA4 Health Cardinoff Grant Ener-LIGHT; European Union Horizon 2020 research and innovation programmes Oncobiome (grant agreement number: 825410, Project Acronym: ONCOBIOME), Prevalung (grant agreement number 101095604, Project Acronym: PREVALUNG EU), and Neutrocure (grant agreement number 861878: Project Acronym: Neutrocure); National support managed by the 10.13039/501100001665Agence Nationale de la Recherche under the France 2030 program (reference number 21-ESRE-0028, ESR/Equipex+ Onco-Pheno-Screen); Hevolution Network on Senescence in Aging (reference HF-E Einstein Network); 10.13039/501100006364Institut National Du Cancer (INCa); 10.13039/501100004795Institut Universitaire de France; 10.13039/501100018872Labex Immuno-Oncology ANR-18-IDEX-0001; PAIR-Obésité INCa_1873, the RHUs Immunolife and LUCA-pi (ANR-21-RHUS-0017 and ANR-23-RHUS-0010, both dedicated to France Relance 2030); and 10.13039/100015545Seerave Foundation; SIRIC Cancer Research and Personalized Medicine (CARPEM, SIRIC CARPEM INCa-DGOS-Inserm-ITMO Cancer_18006 supported by 10.13039/501100006364Institut National Du Cancer, 10.13039/501100014630Ministère des Solidarités et de la Santé and 10.13039/501100001677Inserm). This study contributes to the IdEx Université de Paris Cité ANR-18-IDEX-0001. P.L. is supported by 10.13039/501100006364Institut National Du Cancer (Inca_20195) and 2025 PHC Cai Yuanpei project. O.K. receives funding by 10.13039/100007391Association pour la Recherche sur le Cancer (ARC) and 10.13039/501100006364Institut National Du Cancer (INCa). Y.M. is supported by Brain Science and Brain-like Intelligence Technology-National Science and Technology Major Project (2022ZD0205700), 10.13039/501100003345CAMS Innovation Fund for Medical Sciences (2025-I2M-XHJC-052, 2024-I2M-TS-031, 2021-I2M-1-074, 2023-I2M-2-010, 2022-I2M-2-004), Key Science and Technology Program of Jiangsu Province (BG2025059), Macau Science and Technology Development Fund (FDCT, 0001/2025/AKP), Beijing Research Ward Excellence Program (BRWEP2024W114060100), Suzhou Municipal Key Laboratory (SZS2023005), 2025 PHC Cai Yuanpei project, and the NCTIB Fund for R&D platform for Cell and Gene Therapy. We acknowledge the help of Olivia Bawa and Nicolas Signolle from the PETRA platform, the Imaging and Cytometry platform of the Gustave Roussy Institute and Georges Zadigue from the CFE facility of the Center de Recherche des Cordeliers for technical support. Parts of the figures have been created with licensed version of BioRender.com.

Author contributions

P.L., G.K., and O.K. conceived and supervised the study; G.K. wrote the first draft of the paper with the help of P.L., Y.P., L. Zhao, P.L., J.L., M.M., A.-L.T., J.L.N., S.A.S., H.P., F.D., M.V., and H.F. conducted experiments. D.M. performed molecular modeling. M.C.M., R.J., Y.M., and L. Zitvogel provided conceptual suggestions. All authors revised the manuscript.

Declaration of interests

O.K. and G.K. have been holding research contracts with Daiichi Sankyo, Eleor, Kaleido, Lytix Pharma, PharmaMar, Osasuna Therapeutics, Samsara Therapeutics, Sanofi, Sutro, Tollys, and Vascage. G.K. is on the Board of Directors of the Bristol Myers Squibb Foundation France. O.K. is a scientific co-founder of Samsara Therapeutics. G.K. is a scientific co-founder of everImmune, Osasuna Therapeutics, Samsara Therapeutics, and Therafast Bio. G.K. is in the scientific advisory boards of Hevolution, Institut Servier, Longevity Vision Funds, and Rejuveron Life Sciences. G.K. is the inventor of patents covering therapeutic targeting of aging, cancer, cystic fibrosis, and metabolic disorders. G.K.’s wife, Laurence Zitvogel, has held research contracts with Glaxo Smyth Kline, Incyte, Lytix, Kaleido, Innovate Pharma, Daiichi Sankyo, Pilege, Merus, Transgene, 9 m, Tusk, and Roche, was on the Board of Directors of Transgene, is a cofounder of everImmune, and holds patents covering the treatment of cancer and the therapeutic manipulation of the microbiota. G.K.’s brother, Romano Kroemer, was an employee of Sanofi and now consults for Boehringer-Ingelheim. The funders had no role in the design of the study; in the writing of the manuscript, or in the decision to publish the results.

STAR★Methods

Key resources table

REAGENT or RESOURCESOURCEIDENTIFIERAntibodiesαβ-actin (HRP)BiolegendCat# 664804αERK1/2BiolegendCat# 686902αERK1/2 Phospho (Thr202/Tyr204)BiolegendCat# 369502αMKK3 Phospho (Ser189/Ser207)Cell Signaling TechnologyCat# 12280αMAPKAPK2 (MK2)Life TechnologiesMA551342αMAPKAPK2 Phospho (Thr334)Life TechnologiesMA528081αPD-1(blockade)BioXcellCat# BE0273Rat IgG2aBioXcellCat# BE0089αIL1βBiolegendCat# 503502Biotin-conjugated αIL1βBiolegendCat# 515801αIL2BiolegendCat# 503702Biotin-conjugated αIL2BiolegendCat# 503804αIL12(p70)BiolegendCat# 511802Biotin-conjugated αIL12(p70)BiolegendCat# 505302αTNFαBiolegendCat# 506102Biotin-conjugated αTNFαBiolegendCat# 516003αCD3_BUV737BD BioscienceCat# 612803αCD4_BUV496BD BioscienceCat# 612952αFoxp3_FITCBD BioscienceCat# 567453αIL17A_APC/Cy7BD BioscienceCat# 560821αCD3_PerCP/Cy5.5Life TechnologiesCat# 45-0031-82αCD8_PerCP/Cy5.5Life TechnologiesCat# 45-0081-82αCD44_APCLife TechnologiesCat# 17-0441-82αCD62L Alexa Fluor 700Life TechnologiesCat# 56-0621-82αCD69_eFluor 450Life TechnologiesCat# 48-0691-82αGranzyme B-FITCLife TechnologiesCat# 11-8898-82αIL2_PE/Cy7Life TechnologiesCat# 25-7021-82αIFNγ_PELife TechnologiesCat# 12-7311-82αTNFα_APCLife TechnologiesCat# 17-7321-82αKi-67_eFluor 450Life TechnologiesCat# 48-5698-82αKi-67-BV650Life TechnologiesCat# 416-5698-82αCD45_BV786Life TechnologiesCat# 417-0451-82αCD8_APC/Fire™ 750BiolegendCat# 100766αCD25_BV650BiolegendCat# 102038αCD45_Alexa Fluor 700BiolegendCat# 103128αCD45_APCBiolegendCat# 103112αCD45.1_PE/Cy7Cat# 110730αICOS_PECat# 117406αCTLA4_PE/Cy7Cat# 106314αPD-1_BV786Cat# 135225Bacterial and virus strainsEdit-R lentiviral CAG-Blast-Cas9 nuclease particlesHorizon DiscoveryCat# VCAS10129pCDH-lenti-FPR1_WTThis paperN/ApCDH-lenti-FPR1_T360EThis paperN/ApCDH-lenti-Empty vectorThis paperN/ApCDH-lenti-ZsGreen-OVAThis paperN/ABiological samplesHealthy adult peripheral blood mononuclear cells (PBMCs)CTIBiotech FranceCat# CTICC.IMPB.1Chemicals, peptides, and recombinant proteinsβ-mercaptoethanolSigma-AldrichCat# M3148DexamethasoneSigma-AldrichCat# D0700000Doxycycline hyclateSigma-AldrichCat# D3000000Recombinant murine GM-CSFMiltenyi BiotecCat# 130-095-735SCREEN-WELL® ICCB Known Bioactives libraryEnzo Life SciencesCat# BML-2840MAPK Compound LibraryMedChemExpressCat# HY-L010; library ID: HY-LD-000005319SB203580MedChemExpressCat# HY-10256SB239063MedChemExpressCat# HY-11068Skepinone-LMedChemExpressCat# HY-15300PamapimodMedChemExpressCat# HY-10405DoramapimodMedChemExpressCat# HY-10320RalimetinibMedChemExpressCat# HY-13241LosmapimodMedChemExpressCat# HY-10402PD98059MedChemExpressCat# HY-12028Cyclosporin HMedChemExpressCat# HY-P1122HCH6-1MedChemExpressCat# HY-101283BumetanideMedChemExpressCat# HY-17468DDR1-IN-1MedChemExpressCat# HY-13979DDR1-IN-2MedChemExpressCat# HY-U00444N-Formyl-Met-Leu-Phe (FMLP)Sigma-AldrichCat# 47729-10MG-FERK Activation Inhibitor Peptide I, Cell-PermeableSigma-AldrichCat# 328000Ovalbumin (OVA)Sigma-AldrichCat# A5503Oxaliplatin (OXA)Sigma-AldrichCat# Y0000271Mitoxantrone dihydrochloride (MTX)Sigma-AldrichCat# M6545cyclophosphamide (CTX)Sigma-AldrichCat# C3250000Azoxymethane (AOM)Sigma-AldrichCat# A5486dextran sulfate sodium (DSS)Life TechnologiesCat# J63606.22CFSEBiolegendCat# 423801Collagenase type IVLife TechnologiesCat# 17104019DNAse ISigma-AldrichCat# DN25D-BiotinSigma-AldrichCat# B4639Recombinant human GM-CSFMiltenyi BiotecCat# 130-093-867Recombinant Human IFN-α2BiolegendCat# 592706Human Sera Type AB, Heat-inactivatedInstitut de Biotechnologies Jacques BoyCat# 201021334Critical commercial assaysPKH26 staining kitSigma-AldrichCat# PKH26GLDharmaFECT #1 transfection reagentHorizon DiscoveryCat# T-2001-04DharmaFECT Duo transfection reagentHorizon DiscoveryCat# T-2010-03MycoStrip™ Mycoplasma Detection KitInvivoGenCat# rep-mysnc-100CellTracker blueLife TechnologiesCat# C2110CellTracker redLife TechnologiesCat# C34552CellTracker greenLife TechnologiesCat# C2925CellTracker deep redLife TechnologiesCat# C34565LIVE/DEAD fixable yellow dyeLife TechnologiesCat# L34959PureLink™ Genomic DNA Mini KitLife TechnologiesCat# K182002DreamTaq Green PCR Master MixLife TechnologiesCat# K10825× ELISA coating bufferBiolegendCat# 4217011-Step™ Ultra TMB-ELISA substrat solutionLife TechnologiesCat# 34028RNeasy Plus Mini KitQiagenCat# 74134NuPAGE™ LDS Sample Buffer (4×)Life TechnologiesCat# NP00074–12% NuPAGE® Bis-Tris gelsLife TechnologiesCat# NP0336BOXNuPAGE™ MES SDS Running Buffer (20×)Life TechnologiesCat# NP000202Trans-Blot Turbo RTA Mini 0.2 μm PVDF Transfer KitBioRadCat# 1704272ECL start Western Blotting Detection ReagentCytivaCat# RPN3244Mouse IFN-γ ELISPOT SetBD BioscienceCat# 551083Streptavidin-alkaline phosphatase and BCIP/NBT substrateBD BioscienceCat# 551951MojoSort™ Mouse CD8 Naive T cell Isolation KitBiolegendCat# 480044Cell Stimulation CocktailLife TechnologiesCat# 00-4975-03LIVE/DEAD™ Fixable Yellow Dead Cell Stain KitLife TechnologiesCat# L34968Foxp3/Transcription Factor Staining Buffer SetLife TechnologiesCat# 00-5523-00H2Kb-OVA monomerBiolegendCat# 280051Streptavidin-PEBiolegendCat# 405204DNeasy Blood & Tissue KitQiagenCat# 69506TaqMan™ SNP Genotyping Assay human:3266374_1_FPR2, FPR1, rs867228Life TechnologiesCat# 4351379TaqMan™ Genotyping Master MixLife TechnologiesCat# 4371353MojoSort™ Human CD14^+^ Monocytes Isolation KitBiolegendCat# 480048Deposited dataBulk RNA-Seq: Raw and analyzed dataThis paperENA: PRJEB102824; https://www.ebi.ac.uk/ena/browser/view/PRJEB102824Experimental models: Cell linesMurine: Immortalized dendritic cells (iniDCs)Richter C et al.68N/AMurine: IniDCs stably expressing CRISPR Cas9This paperN/AMurine: IniDCs Fpr1^−/−^This paperN/AMurine: IniDCs Fpr^T360E/T360E^This paperN/AMurine: IniDCs B2m^−/−^This paperN/AMurine: B3ZKarttunen et al.69N/AMurine: MCA205Sigma-AldrichCat# SCC173Murine: MC38Sigma-AldrichCat# SCC172Murine: TC1_LucLin et al.70N/AMurine: OP9ATCCCat# CRL-2749Human: U2OSATCCCat# HTB-96Murine: MCA205 ZsGreen-OVAThis paperN/AHuman: U2OS ZsGreen-OVAThis paperN/AExperimental models: Organisms/strainsMouse: C57BL/6JOlaHsdEnvigoCat# 5705FMouse: OT-I:C57BL/6-Tg(TcraTcrb)1100Mjb/CrlCharles River LaboratoriesCat# 642OT1Mouse: Fpr1^−/−^:C57BL/6NTac-Fpr1^tm1Gao^ N6TaconicCat# 4169Mouse: Fpr1^T360E^: C57BL/6JThis paperN/AOligonucleotidesEdit-R crRNA Non-targeting ControlHorizon DiscoveryCat# U-007501-01-2EGFP Cas9 nuclease mRNAHorizon DiscoveryCat# CAS12217Trans-activating CRISPR RNA (tracrRNA)Horizon DiscoveryCat# U-002005-5000Standard PCR primersThis paper, Table S1N/ASanger sequencing primersThis paper, Table S1N/AqPCR primersThis paper, Table S1N/ASynthetic CRISPR crRNAsThis paper, Table S1N/AHomology template for CRISPR KI mutation of Fpr1 T360EThis paper, Table S1N/ASoftware and algorithmsFiji (ImageJ)Schindelin et al.71https://fiji.sc/Image StitchingPreibisch et al.72https://imagej.net/plugins/image-stitchingTrackMateErshov et al.73https://imagej.net/plugins/trackmate/Graphpad PrismGraphpadhttps://www.graphpad.com/RaNA-seqPrieto et al.74https://ranaseq.eu/iDEPGe et al.75http://bioinformatics.sdstate.edu/idep96/TumGrowthEnot et al.76https://kroemerlab.shinyapps.io/TumGrowth/FlowJoBD Biosciencehttps://www.flowjo.com/QuPath-0.5.0Bankhead et al.77https://qupath.github.io/CytExpert Software for the CytoFLEX PlatformBeckman Coulterhttps://www.beckman.fr/en/flow-cytometry/research-flow-cytometers/cytoflex/softwareImageQuant™ TL analysis softwareCytivaCat# 29800681OtherRPMI 1640 mediumLife TechnologiesCat# 61870010DMEM mediumLife TechnologiesCat# 10566016MEM α mediumLife TechnologiesCat# 22571038HEPES bufferLife TechnologiesCat# 15630056Sodium pyruvateLife TechnologiesCat# 11360070Phosphate-buffered saline (PBS)Life TechnologiesCat# 20012027Penicillin-streptomycin solution (10,000 U/mLLife TechnologiesCat# 15140122GlutaMAX™ supplementLife TechnologiesCat# 35050061TrypLE™ Express dissociation reagentLife TechnologiesCat# 12604013Fetal bovine serum (FBS)Sigma-AldrichCat# F752496-well u-bottom CELLSTAR® TC platesCat# 65018096-well CELLCOAT® POLY-D-LYSINE-treated platesGreiner-Bio-OneCat# 65594696-well high-binding assay platesCorningCat# 9018Red Blood Cell (RBC) Lysis Buffer, 10×BiolegendCat# 420302CTL-Test™ mediumImmunospotCat# CTLT-010GlutaMAX™ SupplementLife TechnologiesCat# 35050061RIPA bufferLife TechnologiesCat# 8990110× TBSEUROMEDEXCat# ET220-BTween 20Sigma-AldrichCat# P1379HRP-AvidinBiolegendCat# 405103

Experimental model and study participant details

Human specimens

Human peripheral blood mononuclear cells (PBMCs) were obtained from CTIBiotech (Cat# CTICC.IMPB.1, all from healthy female donors, 22–55 years old) and were thawed as suggested by the supplier’s suggestions. 1/10 of cells were used for genomic DNA extraction (Qiagen DNeasy Blood & Tissue Kit) according to the manufacturer’s instructions. Genotyping of the FPR1 SNP Rs867228 was performed using the TaqMan SNP Genotyping Assay (Cat# 4351379, human:3266374_1_FPR2, FPR1, rs867228) and TaqMan Genotyping Master Mix (Cat# 4371353), both from Applied Biosystems, on a QuantStudio3 real-time PCR instrument. PCR reactions and allele calls were carried out following the supplier’s protocol and all genotype calls were visually inspected and confirmed by triplicates for ambiguous samples. For generation of monocyte-derived DCs (moDCs), CD14^+^ monocytes were purified from the remaining PBMCs by negative selection using magnetic microbeads (Biolegend, Cat# 480048). Purified monocytes were plated in RPMI-1640 supplemented with 10% human serum Type AB, 1% penicillin/streptomycin, recombinant human GM-CSF (50 ng/mL) and recombinant human IFN-α2b (2,500 U/mL) and cultured at 37°C/5% CO_2_ for 72 h to obtain human monocyte-derived DCs (moDCs). The obtained moDCs were harvested, counted and stained with CellTracker dyes prior to treatment with SB203580 or skepinone-L. In vitro migration was performed as described with murine de-iniDCs, yet using an OVA-expressing U2OS osteosarcoma cells that were killed with oxaliplatin.

Mice

All wild type C57BL/6J mice (6–8 weeks of age) were purchased from Envigo Rms Sarl (Gannat, France) and maintained under specific pathogen-free (SPF) conditions with environmental control, a 12-h light/dark cycle, and ad libitum access to food and water. OT1 mice (females, 6 weeks of age) were obtained from Charles River France. Fpr1 knockout mice (Fpr1^−/−^) were originally produced by Taconic (commercial name C57BL/6NTac-Fpr1tm1Gao N6), subsequently bred in house, and used for experiments at 6–8 weeks of age. The Fpr1 mutant mice (Fpr1^T360E^) were generated by OZgene (Perth, Australia) by knocking T360E into exon 2 of the Fpr1 gene via gene targeting in mouse ES cells. The knockin was made by mutation of the Thr codon ACT to the Glu codon GAG. Fpr1^T360E^ mice were subsequently bred in house and used for experiments at 6–8 weeks of age. All animal experiments adhered to EU Directive 63/2010 and ethical protocols: project #44493–2023080405573528 v2 approved by the ethical committee of Gustave Roussy Campus Cancer (CEEA IRCIV/IGR no. 26); projects #24973–2020040413162969 v3, #33357–2021100514122462 v3, #33386–202109281404192 v2, and #51101-2024091616366641v3 approved by the Charles Darwin Ethical Committee (C2EA–05) that are registered with the French Ministry of Research. Each experiment was conducted using mice of a single sex (male or female), determined by breeding availability. Bone marrow reconstitution was performed with sex-matched donors and recipients. No obvious sex-dependent differences were observed.

Bone marrow from WT, Fpr1^−/−^, Fpr1^T360E^ mice was flushed from femurs and tibias, dissociated into single-cell suspensions, and cryopreserved at −80°C (or liquid nitrogen for long-term storage). For transplantation, bone marrow cells were thawed in a 37°C water bath, washed with warm PBS supplemented with 5% FBS, and resuspended in cold PBS. Lethally irradiated (10 Gy) congenic recipient mice received 5×10^6^ bone marrow cells intravenously (i.v.) and were monitored for at least 6 weeks post-transplantation before tumor model establishment.

Orthotopic fibrosarcoma, colorectal carcinoma, and NSCLC models were created as described previously.78 For the fibrosarcoma model, 5×10^5^ wild-type MCA205 cells or 1×10^6^ wild-type MC38 cells were subcutaneously injected into the right flank. Mice were randomized into treatment groups once tumors became palpable, with tumor surface areas (∼20–25 mm^2^) calculated as longest dimension × perpendicular dimension×π/4. Tumors exceeding 250 mm^2^ resulted in euthanasia.

For the TC1 NSCLC model, 5×10^5^ TC1-Luc cells in 100 μL PBS were i.v. injected. Tumor development was monitored via in vivo bioluminescence imaging of luciferase activity. Tumor incidence was confirmed with a 4-min imaging exposure (6–7 days post-injection), after which mice were randomized into treatment groups. Bioluminescence imaging involved intraperitoneal (i.p.) injection of 3 mg beetle luciferin potassium salt in DPBS (Promega, Madison, WI, USA), followed by imaging using the IVIS Lumina III system (Caliper Life Sciences Inc., Hopkinton, MA, USA). Photon acquisition started with 4-min exposures, reducing to 1 min upon signal saturation. Mice with photon saturation at 1-min exposure using small binning settings were euthanized. Tumor size and progression were assessed frequently.

For the inflammation-induced colorectal/ileum oncogenesis, the bone marrow of female C57BL/6J wild-type mice were reconstituted with that of donors carrying the WT, knockout, or T360E mutation of Fpr1. Mice were let to recover for 7 weeks and were then cohoused for one week to homogenize microbiome. The induction of colon/ileum carcinogenesis was performed as previously described,14 In brief, mice received a single intraperitoneal (i.p.) injection of azoxymethane (AOM, 10 mg/kg) at day 0, and hree days post-AOM injection, mice were provided 2% dextran sulfate sodium (DSS) in their drinking water for 4 days. This was followed by a two-week recovery period without DSS. The cycle of DSS administration and recovery was repeated twice more, resulting in a total of three DSS exposure cycles interspersed with recovery phases. Mice were sacrificed 10 weeks after AOM injection via cervical dislocation. After euthanasia, the ileum and colon were carefully dissected, opened longitudinally, and rinsed with PBS, and rolled from the proximal to distal end to form Swiss rolls. These rolls were pinned in place, fixed in 4% formaldehyde at 4°C for 24 h, and transferred to 70% ethanol for storage prior to paraffin embedding. Subsequently, hematoxylin, eosin, and saffron (HES) staining were performed, and slides were scanned at a resolution of 0.22 μm per pixel by the Experimental Pathology Platform (PETRA) in Gustave Roussy. Slides were analyzed with QuPath-0.5.0 software (https://qupath.github.io/). Lesions were categorized following internationally accepted guidelines and terminology for mouse pathology.79^,^80 To ensure unbiased results, sample identity was blinded during evaluation, which was conducted independently by two researchers.

Adoptive DC transfers, vaccinations, and in vivo detection

IniDC_Cas9 cells or gene-KO derivatives were cultured in medium without Dex/Dox for three days to differentiate into de-iniDCs. For i.v. injection as cancer therapeutic vaccination, tumor antigen exposure was achieved by adding lysates prepared from equal numbers TC1-Luc cells (generated through repeated freeze–thaw cycles and sonication) to the culture for 2 h; while for subcutaneous (s.c.) vaccinating tumor-free mice, the de-iniDCs were exposed to soluble OVA (2 mg/10^6^ DCs) overnight. For some experiments, de-iniDCs were pretreated with MAPK p38α inhibitors (10 μM) for 4 h prior to antigen exposure. Then, antigen-loaded cells were harvested via trypsinization or scraping with a cell lifter, washed twice with cold PBS, and passed through a 70 μm strainer to ensure a single-cell suspension. Processed de-iniDCs were resuspended in cold PBS for i.v. injection (200 μL containing 2×10^6^ cells/mouse) into lung cancer mice, or s.c. injection into (100 μL containing 2×10^6^ cells/mouse) into the flank of hair-shaved naive mice. For histological analysis post-injection, lungs were excised with the trachea, flushed with cold PBS, and fixed by infusing 4% PFA through the trachea, followed by sealing with surgical sutures. The fixed lungs were immersed in 4% PFA before paraffin embedding, sectioning, and subsequent immunohistochemical detection of Cas9-expressing de-iniDCs. Alternatively, to assess DC trafficking in vivo, Fpr1 WT, KO, and T360E mutant de-iniDCs were differentially labeled with CellTracker dyes (blue for WT, green for KO, and deep red for mutant) prior to treatment with the MAPK p38α inhibitor SB203580 (10 μM, 4 h) and subsequent stimulation with TC1 tumor cell lysates (16 h). Following stimulation, the three DC populations were mixed at a 1:1:1 ratio and intravenously injected into TC1 tumor–bearing mice. At 24 h post-injection, lungs were harvested, mechanically dissociated, and subjected to red blood cell lysis before preparation of single-cell suspensions. Cells were stained with a live/dead yellow dye and anti-CD45 antibody, fixed, and analyzed by flow cytometry to quantify the relative presence of each DC subset in the lung. Cell abundance was determined as their percentage in all viable CD45^+^ cells.

OVA-specific T cell activation in vivo

Wild-type and Fpr1^T360E^ mutant C57BL/6 mice were randomly assigned to treatment arms and either received solvent or SB203580 (i.p. at day0 and re-dose 3 days later) prior to vaccination. Vaccination was performed by s.c. injection of OVA-expressing MCA205 cells that had been pretreated in vitro with oxaliplatin to induce immunogenic cell death (cells washed and injected at 1×10^6^ cells per mouse). Seven days after vaccination mice were euthanized and blood and spleens were harvested; splenocytes were prepared by mechanical disruption through a 70 μm mesh and red blood cells were lysed, while peripheral blood mononuclear cells were isolated by standard RBC lysis. Single-cell suspensions were counted and resuspended in serum-free CTL-Test medium (Immunospot, Cat# CTLT-010) supplemented with 1× GlutaMax, 100 U/mL penicillin + streptomycin (Gibco) and seeded in 96-well PVDF ELISPOT plate (5×10^5^/well in triplicate) pre-coated with anti-murine IFN-γ antibody from the ELISPOT Mouse IFN-γ ELISPOT Set (BD, Cat# 551083). Cells were stimulated with OVA257-264 (SIINFEKL) peptide at 1 μg/mL for 16 h and then plates were developed using biotinylated anti–IFN-γ detection antibody, streptavidin-alkaline phosphatase and BCIP/NBT substrate (BD, Cat# 551951) according to the manufacturer’s instructions. Plates were air-dried and spots enumerated using an automated ELISPOT reader; responses are reported as mean spot count ±SEM and analyzed across genotypes and treatment groups.

OT1 transfer and CFSE dilution assay

Naive CD8^+^ T cells were purified from the spleen, peripheral blood, and lymph nodes (inguinal+ axillary+ mesenteric) of OT-I transgenic mice after mechanical dissociation and red blood cell lysis, followed by negative selection using magnetic beads (Biolegend Cat# 480044). Purified OT-I cells were labeled with 5 μM CFSE (Biolegend Cat# 423801) for 10 min at 37°C and quenched with ice-cold RPMI containing 10% FBS. After washing, 1×10^6^ CFSE-labeled OT-I cells were adoptively transferred intravenously into recipient Fpr1 WT or T360E mutant mice, which had been pretreated with solvent or SB203580 as indicated. Mice were then vaccinated subcutaneously with oxaliplatin-pretreated OVA-expressing MCA205 cells (1×10^6^ per mouse). Seven days later, spleens were harvested from recipient mice, processed into single-cell suspensions, and stained with CD8 and CD45.1 to distinguish donor from host T cells. Proliferation of OT-I donor cells was evaluated by CFSE dilution using flow cytometry, and results were expressed as the percentage of divided cells or as proliferation index calculated in FlowJo.

Chemical formulations and administration

SB203580 and bumetanide were dissolved in a solvent comprising 10% Tween-80, 10% PEG400, and 4% DMSO in physiological saline. OXA, MTX, and CTX were dissolved in PBS. These compounds were administered (i.p.) at a dose of 10 or 25 mg/kg for SB203580, 10 mg/Kg for OXA, 5.17 mg/kg for MTX, and 50 mg/kg for CTX, according to the schedules specified in the figures and legends. For checkpoint blockade therapy, mice received i.p. injections of 200 μg anti-PD-1 antibody or 200 μg isotype control antibody according to the schedules specified in the figures and figure legends.

Tissue dissociation and flow cytometry staining

Sample preparation

For the assessment of T cell activation in orthotopic TC1 NSCLC tumor bearing mice adoptively transferred with de-iniDCs, animals were sacrificed at day 3 post DC transfer. Total blood was collected via cardiac puncture under isoflurane anesthesia into 2 mL EDTA-K tubes. Mice were subsequently euthanized, and tumor-bearing lungs, along with the mediastinal lymph nodes (lung tumor-draining lymph node, tdLN) and the spleen were harvested. Samples were maintained on ice in cold RPMI 1640 medium until processing. For the evaluation of de-iniDC antigen cross presentation in tumor-free mice, the blood, spleen, and vaccine-draining lymph nodes were collected following the same procedure.

Dissociation protocol

Total blood was centrifuged at 2000 g to sediment blood cells. After removing the plasma supernatant, blood cells were treated with 1× red blood cell lysing buffer (RBC lysing buffer, BioLegend) to remove erythrocytes. RBC lysing was stopped 2 min later by mixing with 10 times volume of PBS, passing through 70 μm strainers to remove coagulates, and spun down to collect white blood cells. Spleen and lymph nodes were mechanically dissociated through 70 μm strainers using a syringe’s rubber tip to create single-cell suspensions, washed with cold PBS and spun down to collect immune cells. Splenocytes were further treated with RBC lysing buffer to remove erythrocytes. Tumor-bearing lungs were digested enzymatically in a buffer containing 1 mg/mL collagenase type IV (Life Technologies) and DNase I (Sigma) for 1.5 h at 37°C. Dissociated cells were filtered through 70 μm strainers, washed twice with cold PBS, and treated with RBC lysing buffer. The collected immune cells were either directly subjected to antibody staining as described below, or seeded in 96-well U-shape suspending cell culture plates and stimulated with the T cell stimulations. Immune cells isolated from TC1 lung cancer-bearing mice, the cells were incubated with 1× Cell Stimulation Cocktail (plus protein transport inhibitors, Cat# 00-4975-03, eBiscience) for 6 h to detect cytokine production by flow cytometry; and for the cells isolated from mice vaccinated with OVA-pulsed de-iniDCs were stimulated for 24 h with the OVA peptide (257–264, SIINFEKL) at a final concentration of 5 μg/mL.

Immunofluorescent staining

Cells were collected, washed once with cold PBS, and labeled with LIVE/DEAD fixable yellow dye to exclude damaged or dead cells. The CD16/CD32 antibodies diluted in FACS buffer (1% BSA in PBS) were directly added to block nonspecific Fc receptor binding. After centrifugation and discarding of supernatant, cells were stained with a mixture of fluorescence-conjugated antibodies targeting the surface-expressing markers at 4°C in the dark for 30 min. After surface labeling, cells were permeabilized and fixed using a Foxp3/Transcription Factor Staining Buffer kit (Life Technologies), followed by staining with fluorescence-conjugated FOXP3 antibody, Ki-67 antibody, and/or the antibodies targeting intracellular accumulated cytokines, at 4°C in the dark for 30 min. Stained cells were then spun down ad washed twice with the 1× permeabilization buffer provided in the Foxp3/Transcription Factor Staining Buffer kit, and finally resuspended in FACS buffer for analysis. Samples were stored at 4°C until data acquisition.

For the detection of OVA-TCR expressing T cells, the H2-Kb-OVA tetramer was assembled according to the Biolegend protocols: For 15 tests, 30 μL of H2Kb-OVA monomer (Cat# 280051) was mixed with 3.3 μL of streptavidin-PE (Cat# 405204), pipette to mix, and incubate on ice in the dark for 30 min. During the incubation, blocking solution was prepared by combining 80 μL of 1 mM D-Biotin (Cat# B4639, Sigma, diluted in PBS) and 6 μL of 10% (w/v) NaN_3_ with 114 μL PBS. After the incubation, 2.4 μL of blocking solution was added to stop the reaction, which was further incubated at 2°C–8°C overnight. Before use, assembled tetramers were centrifuged at 2500×g for 5 min at 4°C and stored on ice in the dark. For tetramer staining, the OVA-SIINFEKL peptide-stimulated immune cells were washed with cold PBS and stained with a LIVE/DEAD fixable yellow dye for 25 min, followed by incubation with a 1/100 dilution of H2Kb-OVA tetramer in FACS buffer (200 μL/sample) at 4°C in the dark for 30 min. After washing, Fc receptors were blocked with a 1/200 Fc block in FACS buffer (50 μL/well) for 10 min. Cells were then spun down, the supernatant discarded, and stained with a mixture of antibodies targeting T cell surface markers (100 μL/well) for 30 min at 4°C in the dark. Following staining, cells were washed twice with 200 μL FACS buffer, fixed in 1% PFA for 20 min, washed twice, and acquired by FACS within 24 h. Flow cytometric data acquisition was using a BD LSRFortessa flow cytometer (BD Biosciences) or CytoFLEX flow cytometer (Beckman Coulter) and analyzed with FlowJo software or the CytoExpert software. Gating strategies used for analysis are detailed in Supplementary Figures.

Cell lines

Parental immortalized dendritic cells (iniDCs) were generously provided by Sebastian Thieme and collaborators.68 IniDCs stably expressing CRISPR Cas9 (iniDC_Cas9) were created by transduction with the Edit-R lentiviral CAG-Blast-Cas9 nuclease particles (Cat# VCAS10129, Horizon Discovery, Waterbeach, UK), followed by single-cell cloning and validation as previously described.19 Additional gene-knockout iniDC_Cas9 cell lines were established by transfecting iniDC_Cas9 cells with specific crRNA and tracrRNA using the DharmaFECT #1 transfection reagent (Cat# T-2001-04, Horizon Discovery), followed by single-cell sorting and validation via sanger sequencing or immunoblotting. The knockout or point mutation of Fpr1 in parental iniDCs were performed by co-transfecting the iniDCs with custom-designed sgRNA, single-strand DNA donor oligos (sequence listed in Table S1), and an EGFP Cas9 nuclease mRNA (Cat# CAS12217, Horizon Discovery), using the DharmaFECT Duo transfection reagent (Cat# T-2010-03, Horizon Discovery). Clones were generated through single-cell sorting based on transient EGFP expression and subsequently genotyped using Sanger sequencing of PCR amplicons, with genomic DNA serving as a template.

The basic DC culture medium consisted of RPMI 1640 supplemented with 10% decomplemented FBS, 1 mM sodium pyruvate, 10 mM HEPES, and 1× penicillin-streptomycin. β-Mercaptoethanol (50 μM) and recombinant GM-CSF (10 ng/mL) were freshly added prior to use. IniDCs and derived cell lines were maintained under dexamethasone and doxycycline treatment (Dex, 100 nM; Dox, 2 μM), induced the expression of the SV40 large T antigen facilitating immortalization. Removal of Dex/Dox halted cell proliferation and triggered differentiation into immature DCs (“de-iniDCs”) used for experiments. The B3Z T cell hybridomas, kindly provided by Sebastian Amigorena, were maintained in DC medium supplemented with β-mercaptoethanol (50 μM). MCA205 fibrosarcoma cells (Cat# SCC173, Sigma-Aldrich), MC38 murine colon adenocarcinoma cells (Cat# SCC172, Sigma-Aldrich), and luciferase-expressing TC1 non-small cell lung cancer cells (TC1_Luc), a kind gift from T.-C. Wu,70 were cultured in DMEM supplemented with 10% decomplemented FBS and 1× penicillin-streptomycin. The fibroblast-like OP9 feeder cells were purchased from ATCC (Cat# CRL-2749), and are cultured in MEM α medium containing 20% decomplemented FBS, 1 mM sodium pyruvate, 10 mM HEPES, 1× GlutaMAX supplement, 1× penicillin-streptomycin, and freshly added β-mercaptoethanol (50 μM). OVA-expressing MCA205 and U2OS cells were generated in the lab. Mycoplasma contamination was regularly monitored using the MycoStrip Mycoplasma Detection Kit (Cat# rep-mysnc-100, InvivoGen, Toulouse, France). Cell lines were maintained for no more than 15 passages post-thaw, and cultured were restarted from cry stocks prepared at the second or third passage, confirmed to be mycoplasma-free.

Method details

Arrayed CRISPR KO screening and stable knockout generation in Cas9-expressing iniDCs

Predesigned synthetic Edit-R crRNA (Table S1), custom designed sgRNA, single strand HDR donor oligos, non-targeting control crRNA (Cat# U-007501-01-2), trans-activating CRISPR RNA (tracrRNA, Cat# U-002005-5000), and Edit-R EGFP Cas9 Nuclease mRNA (Cat# CAS12217), were all obtained from Horizon Discovery. Both crRNA and tracrRNA were prepared as 10 μM stock solutions in Tris buffer (pH 7.4). The crRNA:tracrRNA duplex was co-transfected following established protocols.19 In brief, iniDC_Cas9 cells were seeded into 6-well plates at 1×10^6^ cells per well in 2 mL of DC medium without Dex/Dox. Each transfection was carried out with 25 nM of crRNA and 25 nM of tracrRNA, mixed in 100 μL RPMI 1640 medium and incubated for 5 min at room temperature. In parallel, 10 μL of DharmaFECT #1 transfection reagent was mixed with 100 μL RPMI 1640 medium and incubated for 5 min. The two solutions were then combined and incubated for an additional 20 min before being added dropwise to the cell culture. For the generation of T360E point mutation and knockout of Fpr1 in the parental iniDCs via homology-directed repair (HDR), an Edit-R EGFP Cas9 Nuclease mRNA (5 ng/well) and single strand HDR donor oligos (100 nM/well) were co-transfected with the custom-designed sgRNA by DharmaFECT Duo transfection reagent, following the same procedure as described above. Three days post-transfection, cells were collected for FACS sorting to isolate clones. For genetic screening, the transfection medium was replaced with fresh DC medium, and cells were allowed to recover overnight prior to the in vitro antigen cross-presentation assay.

Genotyping of transfected cells or sorted clones by sanger sequencing

Genomic DNA was extracted from transfected cells or expanded clones using a PureLink Genomic DNA Mini Kit (Cat# K182002, Life Technologies) following the manufacturer’s protocol. PCR amplification of the target genomic region was performed using gene-specific primers designed to flank the edited site (oligo sequences are provided in Table S1. The PCR were performed with the DreamTaq Green PCR Master Mix (Cat# K1082, Life Technologies) in a thermal cycler with the following cycling conditions recommended in the kit. The PCR amplicons were then diluted and submitted for Sanger sequencing using specific sequencing primers (Table S1). Sequencing data were compared with the reference sequence using alignment software (https://www.benchling.com/academic), thus enabling the identification of desired edits or mutations.

In vitro antigen cross presentation assay

Bone marrow-derived dendritic cells (BMDCs), de-iniDCs, and their genetically modified derivatives, or de-iniDCs transiently transfected with crRNA for screening, were harvested by trypsinization and prepared at a density of 5×10^5^ cells/mL in DC medium supplemented with β-mercaptoethanol and GM-CSF. A 100 μL cell suspension (5×10^4^ cells/well) was plated into 96-well U-bottom tissue culture plates. Cells were treated with or without desired compounds as specified in the figure legends. Soluble OVA was added at a final concentration of 1 mg/mL, and the cultures were incubated at 37°C with 5% CO_2_ for 4 h. In some cases, the soluble OVA was replaced with 2 × 10^5^/well of oxaliplatin-treated (300 μM for 24h) OVA-expressing MCA205 cells, or 2 ng/mL OVA SIINFEKL peptide. Following incubation, the plates were centrifuged at 500×g for 5 min, and the supernatant was replaced with 200 μL/well of fresh RPMI 1640 medium. This washing step was repeated to ensure the removal of residual OVA. Separately, B3Z T cell hybridomas were collected by centrifugation and resuspended at 5×10^5^ cells/mL in DC medium. After washing the DCs, 200 μL/well of the B3Z cell suspension was added to the wells, and the DCs and T cells were co-incubated at 37°C for 18 h. At the end of the co-incubation, the plates were centrifuged at 500×g for 5 min, and 150 μL of the supernatant was carefully transferred for IL2 quantification via ELISA.

Customized ELISA

ELISA assays for IL1β, IL2, and IL12(p70), and TNFα were conducted as described previously.19 Briefly, capture antibodies were diluted 1:500 in 1× ELISA coating buffer (prepared from 5× ELISA coating buffer, Cat# 421701, Biolegend) and applied at 100 μL per well in 96-well high-binding assay plates (Cat# 9018, Corning). The plates were incubated overnight at 4°C, washed three times with washing buffer (1× TBS containing 0.1% Tween 20, 300 μL/well), and blocked with 150 μL/well of blocking buffer (10% FBS and 1% BSA in PBS) for 2 h at room temperature to prevent nonspecific binding. Following blocking, the plates were loaded with either sample supernatants or serially diluted standards and incubated for 2 h at room temperature. After incubation, the wells were washed four times with washing buffer. Biotinylated detection antibodies diluted 1:500 in blocking buffer were then added at 100 μL/well, and the plates were incubated for 1 h at room temperature. After washing the plates four more times, 100 μL/well of HRP-Avidin (Biolegend, Cat# 405103, diluted 1:1000 in blocking buffer) was added and incubated for 30 min at room temperature. Following this, the plates were washed five times, and 100 μL/well of 1-Step Ultra TMB-ELISA substrate solution (Life Technologies, Cat# 34028) was added for color development. When the wells containing the highest concentration standard wells turned dark blue (usually within 10 min), the reaction was stopped with 50 μL/well of stopping buffer (0.5 M H_2_SO_4_). Absorbance at 450 nm was immediately measured using a BMG FLUOstar plate reader (BMG Labtech, Ortenberg, Germany). Cytokine concentrations in the samples were determined using the standard curve and adjusted for sample dilution factors.

Bulk RNA-sequencing (RNA-Seq) analysis

Total RNA was isolated from approximately 5×10^7^ cultured de-iniDCs or BMDCs using the RNeasy Plus Mini Kit (Cat# 74134, Qiagen) according to the manufacturer’s instructions. RNA quality control, library preparation and sequencing service were provided by Biomarker Technologies (BMK, Münster, Germany). The acquired fastq.gz raw data was processed via the online tool RaNA-seq74 to generate gene count table and quality control validations. Differential expression and pathway analysis with the count table were performed by means of the online tool iDEP.75 Only genes with a minimum of 0.5 counts per million in at least 1 library were considered for the analysis. The top 2000 most variable genes were included for k-means analysis of pathway enchainment. Differential expression quantification was conducted at the gene level using the DESeq2. Genes were identified as statistically differentially expressed if they exhibited at least a 2-fold-changes with false discovery rate (FDR) below 0.1. Pathway and network enrichment analyses were performed combining results from upregulated, downregulated, and all regulated genes. Pathways were considered significant if the associated p-values were less than 0.05.

Immunoblotting

Immunoblotting was conducted following the standard protocol for the NuPAGE electrophoresis system (Invitrogen), with reagents obtained from Life Technologies unless stated otherwise. Protein extracts were prepared by lysing cells in RIPA buffer supplemented with a protease inhibitor cocktail, 1× SDS Loading Buffer, and 1× Sample Reducing Buffer. Proteins were resolved using 4–12% NuPAGE Bis-Tris gels in NuPAGE MES SDS Running Buffer and transferred onto 0.45 μm polyvinylidene fluoride (PVDF) membranes using the Trans-Blot Turbo Transfer System (Bio-Rad, Hercules, CA, USA). The membranes were blocked for 1 hour at room temperature in 5% BSA prepared in TBST (Tris-buffered saline with 0.05% Tween 20) to prevent nonspecific binding, then incubated overnight at 4°C with primary antibodies. Following five washes with TBST, blots were incubated for 2 hours at room temperature with HRP-conjugated secondary antibodies (Southern Biotechnologies, Birmingham, AL, USA). After five additional washes with TBST, chemiluminescent signals were developed using the Amersham ECL Prime kit and visualized on the High-resolution Amersham ImageQuant 800 western blot imaging systems (Cytiva Life Sciences). Band intensities were quantified using ImageQuant TL software.

Cell staining and fluorescence video microscopy

IniDCs of different genotype were seeded in 12-well plates and let differentiated without Dex/Dox for 3 days, then stained with different CellTracker Dyes (DeepRed for WT de-iniDCs and Red for any other genotype to be compared with WT) diluted in serum-free RMPI1640 medium following the manufacture’s protocol. After staining, the de-iniDCs were subjected to treatments as indicated. The OVA-expressing MCA205 fibrosarcoma cells were seeded in T75 flask, stained with CellTracker blue and subjected to treatment with oxaliplatin (300 μM) overnight to induce immunogenic cell death. The dying MCA205 cells were then collected and resuspended in RMPI medium at a density of 7.5×10^7^ cells/mL), prechilled and mixed with Matrigel matrix (concentration at ∼9 mg/mL total protein) 2:1 to reach a final concentration of 3 mg/mL containing 5×10^7/^mL MCA205 cells. The Matrigel-containing MCA205 cells were loaded in the well center of Poly-D-Lysine-treated 96-well Assay Plate at 1 μL/well to form a semi-spheroid. In the case of using human moDCs, OVA-expressing MCA205 cells were replaced with OVA-expressing human osteosarcoma U2OS cells. Plate was incubated at 37°C for 10 min and then immediately loaded with the OP9 feeder cell suspension (5×10^5^ cells/mL) at 100 μL/well, and let adapt at 37°C for 4 h to form a monolayer. Pre-stained & treated de-iniDCs were collected and diluted to 8×10^4^ cells/mL with DC medium. The DeepRed^+^ WT de-iniDC and Fpr1^−/−^ or Fpr1^T360E^ Red^+^ de-iniDCs were mixed at 1:1 ratio (v/v), and added on top of the OP9 coated plates containing MCA205 semi-spheroids. The plates were immediately subjected to live imaging using an ImageXpressMicro C automated confocal microscope (Molecular Devices, Sunnyvale, California, USA) equipped with a 10× PlanApo objective (Nikon, Tokyo, Japan) and an environmental control system providing optimal cell culture conditions. Images were acquired every 10 min using DAPI, Cy3, and Cy5 filters for a total period of 20 h, with 3 × 3 adjacent imaging sites per well. Time stacked images in tiff format were exported and stitched to whole-well view field with the Stitching plugin72 in ImageJ. Spot detection (representing DCs at each time points) and motility tracking were performed with the TrackMate plugin73 in ImageJ.

Statistical analysis

Statistical analysis was performed using GraphPad Prism (v10) and TumGrowth (GitHub/Kroemerlab),76 employing one-way or two-way ANOVA test (with FDR or Dunnett’s multiple comparisons test), unpaired or paired Student’s t test, Fisher’s exact test, linear or log-transformed mixed-effects models for longitudinal tumor growth comparisons, likelihood ratio tests for endpoint tumor size, and Cox proportional hazards regression or log-rank tests for survival analysis, with significance set at p ≤ 0.05 as annotated in figures.