Articles Macrophage CD5L is a target for cancer immunotherapy Lidia Sanchez-Moral,a,t Tony Paul,a,t Clara Martori,a,b,t Joan Font-Díaz,c Lucía Sanjurjo,a Gemma Aran,a Érica Téllez,a Julià Blanco,d Jorge Carrillo,d Masaoki Ito,e Martina Tuttolomondo,f Henrik J. Ditzel,f ,g Caterina Fumagalli,h Gustavo Tapia,i Julia Sidorova,j Helena Masnou,k,l Marco-Antonio Fernández-Sanmartín,m Juan-José Lozano,j Cristina Vilaplana,n,o,p,q Alhelí Rodriguez-Cortés,b Carolina Armengol,l,r Annabel F. Valledor,c Leonor Kremer,s and Maria-Rosa Sarriasa,l,∗ a Innate Immunity Group, Germans Trias i Pujol Research Institute (IGTP), 08916 Badalona, Spain b Departament de Farmacologia, Terapèutica i Toxicologia, Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain c Department of Cell Biology, Physiology and Immunology, School of Biology, University of Barcelona and Institute of Biomedicine of the University of Barcelona (IBUB), 08028 Barcelona, Spain d Virology and Cellular Immunology (VIC), IrsiCaixa, 08916 Badalona, Spain e Department of Surgical Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, 739-8527 Hiroshima, Japan f Department of Cancer and Inflammation Research, Institute of Molecular Medicine, University of Southern Denmark, 5000 Odense, Denmark g Department of Oncology, Odense University Hospital, 5220 Odense, Denmark h Servicio de Anatomía Patológica, Hospital de la Santa Creu i Sant Pau, 08025, Barcelona, Spain i Pathology Department, Germans Trias i Pujol University Hospital (HUGTiP), 08916 Badalona, Spain j Bioinformatics Platform, CIBERehd, 08036 Barcelona, Spain k Gastroenterology Department, Germans Trias i Pujol University Hospital (HUGTiP), 08916 Badalona, Spain l Network for Biomedical Research in Hepatic and Digestive Diseases (CIBERehd), 28029 Madrid, Spain m Flow Cytometry Unit, Germans Trias i Pujol Research Institute (IGTP), 08916 Badalona, Spain n Experimental Tuberculosis Unit, Germans Trias i Pujol Research Institute (IGTP), 08916 Badalona, Spain o Department of Genetics and Microbiology, Autonomous University of Barcelona, Barcelona, Spain p Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES), 28029 Madrid, Spain q Microbiology Department, Laboratori Clínic Metropolitana Nord, Germans Trias i Pujol University Hospital, 08916 Badalona, Spain r Childhood Liver Oncology Group, Program of Predictive and Personalized Medicine of Cancer (PMPCC), IGTP, 08916 Badalona, Spain s Protein Tools Unit and Department of Immunology and Oncology, Centro Nacional de Biotecnología (CNB-CSIC), 28049 Madrid, Spain Summary eBioMedicine 2023;91: 104555 Background Reprogramming of immunosuppressive tumor-associated macrophages (TAMs) presents an attractive therapeutic strategy in cancer. The aim of this study was to explore the role of macrophage CD5L protein in TAM Published Online xxx https://doi.org/10. activity and assess its potential as a therapeutic target. 1016/j.ebiom.2023. 104555 Methods Monoclonal antibodies (mAbs) against recombinant CD5L were raised by subcutaneous immunization of BALB/c mice. Peripheral blood monocytes were isolated from healthy donors and stimulated with IFN/LPS, IL4, IL10, and conditioned medium (CM) from different cancer cell lines in the presence of anti-CD5L mAb or controls. Subsequently, phenotypic markers, including CD5L, were quantified by flow cytometry, IF and RT-qPCR. Macrophage CD5L protein expression was studied in 55 human papillary lung adenocarcinoma (PAC) samples by IHC and IF. Anti-CD5L mAb and isotype control were administered intraperitoneally into a syngeneic Lewis Lung Carcinoma mouse model and tumor growth was measured. Tumor microenvironment (TME) changes were determined by flow cytometry, IHC, IF, Luminex, RNAseq and RT-qPCR. Findings Cancer cell lines CM induced an immunosuppressive phenotype (increase in CD163, CD206, MERTK, VEGF and CD5L) in cultured macrophages. Accordingly, high TAM expression of CD5L in PAC was associated with poor patient outcome (Log-rank (Mantel–Cox) test p = 0.02). We raised a new anti-CD5L mAb that blocked the immunosuppressive phenotype of macrophages in vitro. Its administration in vivo inhibited tumor progression of lung cancer by altering the intratumoral myeloid cell population profile and CD4+ T-cell exhaustion phenotype, thereby significantly modifying the TME and increasing the inflammatory milieu. *Corresponding author. Innate Immunity Group, Germans Trias i Pujol Research Institute (IGTP), 08916 Badalona, Spain. E-mail address: mrsarrias@igtp.cat (M.-R. Sarrias). t These authors contributed equally to this work. www.thelancet.com Vol 91 May, 2023 1 Articles Interpretation CD5L protein plays a key function in modulating the activity of macrophages and their interactions within the TME, which supports its role as a therapeutic target in cancer immunotherapy. Funding For a full list of funding bodies, please see the Acknowledgements. Copyright © 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Keywords: CD5L; Immunotherapy; Lung adenocarcinoma; Macrophage; Monoclonal antibody; Scavenger receptor cysteine rich Research in context Evidence before this study and enhanced CD5L expression in macrophages. Immunotherapy is aimed at reactivating the immune system Additionally, TAM expression of the protein CD5L in lung to mount a robust antitumoral response. However, although adenocarcinoma human specimens correlated with poor successful in certain cases, current immunotherapy outcomes. We raised a new antibody that specifically binds treatments need improvement. Tumor-associated to CD5L and blocks the immunosuppressive state of macrophages (TAMs) are the most abundant immune cells in macrophages. Administration of this antibody in a mouse tumor. Because they are immunosuppressive in most solid model of cancer modified the TME thereby reducing tumor tumors, TAMs rise as a remarkably promising cell target in growth. immuno-oncology. Implications of all the available evidence Added value of this study Our results point to CD5L as an immunotherapy target for the In this study we report that secreted factors from cancer treatment of lung cancer that may have an important impact derived cell lines induced an immunosuppressive profile in many solid tumors. Introduction Over recent decades, significant advances in patient The tumor microenvironment (TME) is a complex sys- treatment options and outcomes have been achieved in tem comprised of many cell types, including endothelial oncology.12 Specifically, immunotherapy, including cells, smooth muscle cells, fibroblasts, and immune monotherapy and combination therapies, has emerged cells such as macrophages, dendritic cells (DCs), and T as the first-line treatment for many types of tumors.13 lymphocytes.1 Given the critical role of the TME in However, the proportion of cancer patients that can modulating tumor progression and response to treat- benefit from their clinical application is still limited. ment, therapeutic strategies targeting this environment Thus, intense research efforts are underway to identify have recently emerged as a promising approach to tackle other immune-modulatory strategies for cancer. Selec- cancer.2 tive targeting and re-education of immunosuppressive Although immune infiltrates vary depending on the TAMs in the TME is an attractive option that is currently type of cancer, numerous studies have demonstrated being intensively studied.14–16 that tumor-associated macrophages (TAMs) are the In recent years, the contribution of Scavenger Re- major immune component of the tumor stroma.3 Mac- ceptors (SRs) to macrophage activity in the context of rophages are plastic cells that can shift their functional cancer has received increasing attention.17,18 SRs form a phenotypes in response to various microenvironmental large family of proteins predominantly expressed by signals, including those generated by tumor and stromal myeloid cells. They are structurally diverse and partici- cells.4 In this context and depending on their activation pate in a wide range of biological functions, including states, macrophages can exert pro- and anti-tumor endocytosis, phagocytosis, adhesion, and signaling, all functions. In most established progressing mouse and of which ultimately lead to the elimination of degraded human tumors, TAMs usually display an immunosup- or harmful substances.18 Interestingly, the expression of pressive phenotype, geared toward promoting tumor SRs containing cysteine-rich domains (termed SRCR growth and invasiveness directly and via angiogenesis, proteins) such as CD163, SR-A1 and Macrophage Re- tissue remodeling, production of specific cytokines/ ceptor With Collagenous Structure (MARCO) is mark- chemokines, and suppression of adaptive immunity.5–9 edly increased in immunosuppressive macrophages.19 The abundance of these cells correlates negatively with Indeed, CD163 is a widely recognized marker of this survival outcome in several types of solid cancer, phenotype in humans, and its expression is related to including that of the lung10 and liver.8,11 poor prognosis in many types of cancer, including 2 www.thelancet.com Vol 91 May, 2023 Articles breast cancer and lung adenocarcinoma.20,21 In addition, Generation and characterization of RImAb reprogramming TAMs by targeting CD163 and MARCO monoclonal anti-CD5L antibody has been shown to inhibit cancer progression in mouse The generation of murine mAbs against human CD5L models of melanoma, glioma, and colon cancer.22–24 in BALB/c mice was described recently.27 The CSIC CD5-like (CD5L) is a 40-kDa soluble glycoprotein Ethics Committee and the Agriculture Department that belongs to the SRCR superfamily. This protein (Community of Madrid) approved the use of experi- modulates the activity of macrophages in a wide spec- mental animals PROEX 18/14 by Dr. Leonor Kremer for trum of settings, including the pathogenesis of several mAb generation. The 8C4G4D7 mAb was a clone spe- infectious and inflammatory processes.25,26 Also, we cifically selected from a previous hybridoma collection described that CD5L promotes macrophage polarization that we generated by immunizing mice against human towards an immunosuppressive phenotype through CD5L,27 was named RImAb and was produced in cul- enhanced autophagy in vitro.17 However, its specific role ture supernatants and purified by affinity chromatog- in the context of TME macrophages remains unknown. raphy using Protein G-Sepharose (29-0485-81, GE In the present study, we hypothesized that CD5L is a Healthcare, Chicago, IL, USA). The immunoglobulin key player in macrophage responses in the context of subclass was determined by ELISA using specific cancer. We found that CD5L is expressed in TAMs in peroxidase-conjugated antibodies against the heavy human specimens of papillary lung adenocarcinoma (IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM) and light (PAC) and its increased levels are associated with poor (kappa and lambda) chains of mouse immunoglobulins, prognosis. We then generated a CD5L-blocking mono- following the manufacturer’s instructions (37503, clonal antibody (mAb), that when administered in a syn- Thermo Fisher Scientific, Waltham, MA, USA). geneic mouse model of lung cancer reduced tumor Direct binding enzyme-linked immunosorbent assay growth, reprogrammed TAMs, and shifted the anti- (ELISA) was performed by immobilizing 5 μg/ml of inflammatory and pro-tumorigenic TME towards a less recombinant human CD5L (rhCD5L),27 recombinant tumor-permissive environment. In summary, our results mouse CD5L (rmCD5L) (2834-CL-050, R&D systems, point to CD5L as a target for cancer immunotherapy. Minneapolis, MN, USA), human DMBT1 (kindly pro- vided by Dr. Martina Tuttolomondo), and human re- combinant CD5 (1636-CD-050, R&D systems) in 96-well Methods microtiter plates, overnight at 4 ◦ C. Plates were blocked Ethics approval and consent to participate with PBS containing 5% Bovine serum albumin (BSA) All human studies were conducted following the (A4503, Sigma–Aldrich, Saint Louis, MO, USA) for 1 h Declaration of Helsinki principles and current legisla- at room temperature (RT). The mAb was then added to tion on the confidentiality of personal data, and were the wells at the indicated concentrations and incubated approved by the Human Ethics Committee of the for 1 h at RT. Between each step, the plates were washed Germans Trias i Pujol University Hospital (with codes twice with PBS 0.01% Tween-20 to remove unbound PI-20-049 and PI-20-085). Informed consent from all proteins. For bound antibody detection, a 1:5000 dilu- participants (patients providing tumor tissues) was tion of peroxidase (PO)-labeled anti-mouse IgG antibody obtained. (A4416, Sigma–Aldrich, RRID: AB_258167) was added Buffy coats, provided by the Blood and Tissue Bank at the final step and incubated for 30 min at RT. Un- (Barcelona, Spain), were obtained from healthy blood bound antibody was washed three times with PBS donors following the institutional standard operating 0.01% Tween-20. Yellow color was developed by adding procedures for blood donation and processing, 3,30,5,50-tetramethylbenzidine liquid substrate (T8665, including informed consent. Sigma–Aldrich) for 20 min, after which the reaction was Animal care and treatment were carried out in stopped by the addition of 2 M H2SO4, and the optical accordance with Spanish and EU laws. Protocols density was read at 450 nm using a Varioskan Flash requiring animal manipulation were approved by the microplate reader (Thermo Fisher Scientific). Each assay institutional Animal Experimentation Ethics Committee was repeated three times, with similar results. of the Comparative Medicine and Bioimage Centre of Characterization of protein sequence identity and Catalonia (CEEA-CMCiB) (#9455), as well as by animal similarity between human CD5L and human DMBT1, care and use committees of the Parc Científic de Bar- human CD5, and mouse CD5L was calculated using the celona (#9672), Universitat de Barcelona (#7088), and UniProtKB database (NIH, EMBL-EBI), BlastP (NIH) the Government of Catalonia (CEA-OH/10300/1). and SIAS (Sequence Identity and Similarity software, Universidad Complutense de Madrid). Human specimens of PAC Human specimens of PAC were provided by Dr. Immunohistochemistry (IHC) Masaoki Ito. The samples were obtained at the time of Formalin-fixed paraffin-embedded (FFPE) 4 μm tissue surgery and were examined and characterized by expert sections were used for the analysis. IHC staining was pathologists. performed by standard protocol: the sections were www.thelancet.com Vol 91 May, 2023 3 Articles deparaffinized with xylene, followed by rehydration in measurement of stained area percentage. A double graded alcohol concentration. Endogenous peroxidase blinded analysis was performed by two individuals and blocking was performed with 0.3% H2O2 in ethanol the mean value obtained from both evaluators was during the rehydration. Heat-induced antigen retrieval considered for final analysis. Statistical analysis was (HIER) was performed with autoclave using Citrate performed using GraphPad Prism V9. buffer (pH 6, 10×, C-9999, Sigma–Aldrich) at 121 ◦ C for 10 min, followed by permeabilization with 0.2% Tween Immunofluorescence (IF) of FFPE tissue sections in PBS for 10 min. The slides were later incubated with For IF analysis, 4 μm FFPE tissue sections were used primary antibodies dissolved in blocking buffer (3% and processed as per the optimized protocol. Human BSA (A4503-504, Sigma–Aldrich)) +5% donkey/goat samples were studied using the primary antibodies anti- serum, (S30 and S26 respectively, Millipore, Merck, CD5L (1:100) (HPA068384, Sigma–Aldrich, RRID: AB_ Burlington, MA, USA) overnight at 4 ◦ C. For human 2685979) and anti-CD68 (1:100) (Kp-1, 168M-95, Cell samples, IHC analysis was performed using anti-CD5L Marque, RRID: AB_1158188). For mouse samples, antibody (1:100) (HPA068384, Sigma–Aldrich, RRID: anti-CD5L (1:100) (AF2834, R&D systems, RRID: AB_2685979) and goat anti-rabbit IgG coupled to AB_2076369) and anti-F4/80 (1:100) (AB100790, horseradish peroxidase (1:200) (HAF008, R&D Systems, Abcam, RRID: AB_10675322) were used. Depending on RRID: AB_357235) as secondary antibody. For mouse the primary antibody, donkey anti-rabbit (A-21206, samples, a panel of primary antibodies, namely anti-F4/ RRID: AB_2535792), mouse (A-31570, RRID: AB_ 80 (1:100) (AB100790, RRID: AB_10675322), anti-TNF- 2536180), or goat IgG coupled with Alexa-488 and α (1:100) (AB6671, RRID: AB_305641), anti-MPO Alexa-555 (A-21432, RRID: AB_2535853), all from (1:500) (AB45977, RRID: AB_944318), anti-Arg-1 Thermo Fisher Scientific, were used as secondary anti- (1:200) (AB92274, RRID: AB_10563668), anti-iNOS bodies at a dilution of 1:100. Finally, nuclei were stained (1:100) (AB15323, RRID: AB_301857), all from Abcam with PBS containing Hoechst 33258 (1:20000, 94403, (Cambridge, UK), anti-CD31 (1:100) (NB100-2284, Sigma–Aldrich) for 10 min at RT and observed under Novus Biologicals, RRID: AB_577761) and anti-BCL2 the microscope (Leica DMI 6000 B). For IHC and IF, the (1:100) (JF104-8, Thermo Fisher Scientific, RRID: AB_ samples were visualized under 40× magnification and a 2898964), were used with goat anti-rabbit IgG-HRP minimum of five random areas were chosen per sam- (1:200) (AB6721, Abcam, RRID:AB_955447) and donkey ple. The acquired images were analyzed using Image J. anti-goat IgG-HRP (1:500) (AB_2340390, Jackson A multichannel composite image was created using ImmunoResearch, RRID: AB_2340390) as secondary channel tools, separating the three channels (red, green antibodies (30 min incubation). Chromogenic detection and blue) respectively. Color balance for the individual of antibody binding was performed using a DAB sub- channel was performed to adjust the brightness and strate kit (34002, Thermo Fisher Scientific) for 20 min, contrast, eliminating possible background and non- protected from light. Counterstaining was done with specific staining. The split composite images were hematoxylin (1051740500, HX 86017674, Merck). The later merged for a uniform image with which further slides were later mounted with DPX (DPX 06522, analysis and quantification was performed. Statistical Sigma–Aldrich), dried, and observed under a Leica DMI analysis was performed using GraphPad Prism V9. 6000 B microscope (Leica Microsystems, Wetzlar, Germany). Cell culture Microscope Koehler illumination was set up to ach- Peripheral blood mononuclear cells (PBMCs) were iso- ieve the best image resolution and image quality, as lated from leukocyte concentrates from healthy blood recommended by Leica microsystems. The focused donors, as previously described,28 by Ficoll–Paque (17- areas were subjected to contrast and brightness adjust- 1440, GE Healthcare) density gradient centrifugation at ments using Leica software and once the optimum 400g for 20 min after CD3+ cell depletion by RosetteSep conditions were achieved, the images were acquired. human CD3 depletion cocktail (15621, StemCell Tech- For quantification of human samples, images from five nologies, Vancouver, BC, Canada). Recovered cells were random areas were taken. For statistical analysis, mean washed twice in PBS and counted using a mouse anti- of CD5L positive cells was taken for each sample. In- human CD14 antibody (555397, BD Bioscience, RRID: clusion and exclusion criteria for the characterization of AB_395798) and Perfect-Count microspheres (CYT- human CD5L positive macrophages were designed un- PCM Cytognos, Salamanca, Spain), following the man- der careful supervision of a pathologist. For mice sam- ufacturer’s instructions. 5 × 105 or 1 × 106 CD14+ cells ples, the captured images were analyzed using Image J, per well were seeded in 24- or 6-well plates, respectively, color deconvolution 2 plugin. H DAB was chosen as the and peripheral blood monocytes (PB monocytes) were vector with an image output of 8 bits. The DAB channel isolated by adherence in RPMI supplemented with 10% was selected and the threshold was adjusted against a human AB serum (H4522, Sigma–Aldrich), 1% of white background to the original image, considering the 10000 U/ml penicillin and 10 mg/ml streptomycin (P/S; background and non-specific staining, followed by P0781, Sigma–Aldrich) in a 5% CO2 incubator at 37 ◦ C 4 www.thelancet.com Vol 91 May, 2023 Articles for 30 min. Non-adherent cells were removed, and blockade on IL10-induced activation, RImAb was added at adherent cells were washed twice with PBS and incu- a final concentration of 5 μg/ml 45 min before IL10 bated in RPMI supplemented with 10% heat-inactivated addition to the culture. fetal bovine serum (FBS) (DE14-840E, Lonza, Basel, Switzerland) and 1% P/S for 24 h before the experi- Macrophage activation markers by flow cytometry ments. The percentage of adherent CD14+ cells (PB Flow cytometry analysis of macrophage activation monocytes) routinely obtained was 94.98% ( ± 3.26%). markers was performed as previously described.17 In assays performed with human monocyte-derived Briefly, PB monocytes were cultured in 6-well plates macrophages (HMDMs), PB monocytes were differen- (106 cells/well) in RPMI medium containing 5% FBS tiated by incubation in RPMI supplemented with 10% and the indicated stimuli. After 72 h, macrophages were heat-inactivated FBS and 1% P/S for 7 days before the detached with Accutase (Sigma–Aldrich), washed twice experiments, as previously described.29 in PBS, and incubated with 50 μl of PBS containing 10% HepG2 (RRID: CVCL_0027) and SNU398 (RRID: human AB serum, 2% FBS, and 0.02% NaN3 (blocking CVCL_0077) cells were purchased from ATCC (The buffer) for 30 min on ice. Cells were then incubated American Type Culture Collection; Manassas, VA, with a combination of fluorescently conjugated anti- USA) and Huh7 (RRID: CVCL_0336) cells were pur- human HLA-DR-FITC (555811, RRID: AB_396145), chased from RIKEN BRC Cell Bank. These cell lines CD80-PE-Cy7 (561135, RRID: AB_10561688), CD206- were cultured in EMEM (HepG2), DMEM (Huh7) or PE-CF594 (564063, RRID: AB_2732052), CD23-APC RPMI (SNU398), supplemented with 1% P/S and 10% (558690, RRID: AB_1645456), and CD163-BV711 heat-inactivated FBS. A459 and PC9 cells were kindly (563889, RRID: AB_2738469) mAbs, all from BD Bio- provided by Dr. Rafael Rosell (ICO, IGTP), and were sciences, for 20 min in Brilliant stain buffer (563794, cultured in RPMI supplemented with 1% P/S and 10% BD Biosciences) on ice. They were then rinsed with heat-inactivated FBS. All human cell lines have been washing buffer (PBS containing 2% FBS and 0.02% identified and validated using the AmpFLSTR™ NaN3) and fixed with 1% paraformaldehyde. Flow cyto- Identifiler™ Plus PCR Amplification Kit (A26364, metric acquisition was performed on a BD LSR Fortessa Thermo Fisher Scientific) and the GeneMapper v3.2 instrument using FACSDiva software (BD Biosciences), software (RRID:SCR_014290). The Lewis Lung Carci- with 10000 events acquired for each sample. Data noma (LLC) cell line, 3LL-R, was kindly provided by analysis was performed using FlowJoTM 10.8.1 software Dr. Jo van Ginderachter (VIB, Belgium),30 and (BD Biosciences, RRID: SCR_008520). cultured in RPMI supplemented with 1% P/S, 10% FBS, and 1% Glutamax (13462629, Thermo Fisher Fluorescence microscopy studies on activated Scientific). Characterization of the cell line was per- macrophages formed in.31 This cell line has been used for tumor Fluorescence studies were performed as previously growth experiments within 15 passages after thawing. described17,28: HMDMs (105 cells/well) were plated and Mycoplasma testing was performed and all cell lines incubated with the indicated stimuli for 72 h on Mil- tested negative. licell EZ slides (PEZGS0816, Merck). Cells were fixed For the collection of cancer cell-conditioned media with PBS containing 4% paraformaldehyde (141451, (CM), cells were grown to 90% confluency, then washed Panreac), blocked with PBS containing 0.2% Triton with PBS, and the medium was replaced with medium X-100 (X100, Sigma–Aldrich), 5% FBS, and 5% hu- containing 2% FBS. Twenty-four hours later, the super- man AB serum (blocking buffer), and incubated with natant was collected and centrifuged at 10000 rpm for 5 μg/ml RImAb in blocking buffer overnight at 4 ◦ C. 10 min at 4 ◦ C to remove cellular debris. It was then Cells were subsequently incubated with FITC-anti- aliquoted and stored at −80 ◦ C for subsequent experi- mouse IgG/IgM antibody (555988, BD Biosciences, ments, where it was diluted 1:2 with media containing RRID: AB_396275) for 1 h at RT in blocking buffer. 10% FBS. Between steps, unbound antibodies were removed with 3 washes with PBS. Finally, nuclei were stained In vitro activation of macrophages with PBS containing Hoechst 33258 diluted 1:20000 PB monocytes were activated with IFN/LPS, 50 ng/ml (94403, Sigma–Aldrich) for 10 min at RT. Cells were endotoxin-free IFNγ (Peprotech, 300-02-A, Rocky Hill, NJ, then washed 5 times with PBS, and coverslips were USA) plus 100 ng/ml LPS from E. coli O111:B4 (14391, mounted in Fluoromount media (F4680, Sigma– Sigma–Aldrich); IL4, 40 ng/ml endotoxin-free IL4 (200- Aldrich) and stored at 4 ◦ C. 04-A, Peprotech); IL10, 50 ng/ml endotoxin-free IL10 The slides were examined under an Axio Observer (200-10-A, Peprotech) as previously described17; and CM Z1 DUO LSM 710 confocal system (Carl Zeiss Micro- collected from Huh7, HepG2, SNU398, A459, and PC9 scopy GmbH, Jena, Germany) and analyzed with ZEN cells. Macrophages cultured in growth medium were Black software (Carl Zeiss Microscopy GmbH). CD5L referred to as controls. Some of the reference stimuli data fluorescence intensity was quantified using ZenLite have been published.17 To assess the role of CD5L software (Carl Zeiss Microscopy GmbH). www.thelancet.com Vol 91 May, 2023 5 Articles Syngeneic mouse model (00-5523-00, Invitrogen, Thermo Fisher Scientific), Wild-type C57BL/6 mice (Envigo, Indianapolis, IN, following the manufacturer’s specifications. For T-cell USA) (RRID:MGI:5658887) were injected with 3 × 106 memory and exhaustion characterization, the following LLC cells (3LL-R) in PBS subcutaneously in the right antibodies were used: CD3-BV650, CD4-BB700, CD8- dorsal flanks, as described previously.30 Tumors were V500, CD44-FITC (561859, RRID: AB_10894581), allowed to grow for 7 days, and then 150 μg of RImAb in CCR7-BV421 (562675, RRID: AB_2737716), LAG3-APC PBS was administered intraperitoneally (i.p.) or an equal (562346, RRID: AB_11153127), TIGIT-PE (565168, volume of PBS or IgG2a isotype (BE0085, BioXcell, RRID: AB_2739089), from BD Biosciences, and CTLA4- RRID: AB_1107771) was used as a control (n = 8 mice/ PECy7 (106313, RRID: AB_2564237), PD1-BV785 group), every three days. The number of animals was (135225, RRID: AB_2563680), and TIM3-BV711 calculated as follows: accepting an alpha risk of 0.05 and (134021, RRID: AB_2890691) from Biolegend. Subse- a beta risk of 0.2 in a two-sided test, 8 subjects are quently, cells were washed by centrifugation at 700g for necessary in each group to recognize as statistically 5 min, fixed in 100 μl of 1% paraformaldehyde, and significant a difference greater than or equal to stored at 4 ◦ C for a maximum of 24 h. Data were 450 mm3. The common standard deviation was collected using a BD LSR Fortessa instrument and the assumed to be 300 mm3. A drop-out rate of 10% was FACSDiva software (BD Biosciences), with a minimum anticipated. Mice were randomly allocated to the of 10000 events acquired for each sample (see gating experimental groups. Tumor size was measured strategy in Fig. 5), and analyzed using FlowJo™ Soft- manually with a caliper every day, and tumor volume ware (version 10.8.1, BD Biosciences). was calculated using the formula V = π × (w2 × l)/6 To sort the CD45+CD11b+Ly6C+Ly6Glo cell popula- (width, w, and length, l).32 Mice were euthanized at day tion, we repeated the same procedure but cells were 15. Tumors were excised, and blood was collected for incubated only with the CD45-APC-CY7, CD11b- further analyses. Serum was obtained after blood Alexa647, Ly6G-FITC, and Ly6C-BV421 antibodies and centrifugation at 2000g for 15 min and stored at −80 ◦ C were resuspended in PBS-2% FBS after washing. Cells until analyzed. were immediately sorted using the FACSAriaII cell sorter (BD Biosciences) and collected in PBS-2%FBS. Characterization of intratumoral immune cell Pellets from these cells were frozen at −80 ◦ C. populations by flow cytometry and cell sorting Tumors were minced into small pieces and dissociated Analysis of chemokines by Luminex using the GentleMACS™ Dissociator (130-093-235 For tumor sample preparation, 5 mg of each frozen Miltenyi), following the manufacturer’s instructions. tumor was submerged in 0.5 ml of PBS with the rec- CD45+ cells were isolated by magnetic positive se- ommended concentration of complete Mini Protease lection by using EasySep™ Mouse CD45 Positive Se- Inhibitor Cocktail (11836153001, Roche) and then ho- lection Kit (19945, StemCell). The manufacturer’s mogenized using a Bio-Gen™ PRO200® homogenizer instructions were followed. The CD45+ cell suspension for 30 s and centrifuged at 12000 g for 10 min at 4 ◦ C to was diluted to a concentration of 5 × 106 cells/ml in PBS remove insoluble materials. The supernatants contain- and incubated first with Fc block (rat anti-mouse CD16/ ing the tumor extracts were then quantified using the CD32, BD Biosciences) (1:100 dilution, 30 min, 4 ◦ C). Pierce BCA Protein Assay Kit (23227, Thermo Fisher Cells were then incubated with a combination of fluo- Scientific). Chemokine concentration was measured rescently conjugated anti-mouse mAbs for cell surface using the MILLIPLEX® Magnetic Bead Kit (MCYTO- phenotyping by flow cytometry. CD45-APC-CY7 MAG-10K, Merck) and following the manufacturer’s (557659, BD Biosciences, RRID: AB_396774) was used instructions. For tumor analysis, 10 μg of protein was to verify the purity of the leukocyte population. analyzed, and PBS with protease inhibitor was used as a Myeloid cell lineage antibodies comprised CD11b- matrix. For serum analysis, 12.5 μL of serum diluted in Alexa647 (557659, RRID: AB_396774), Ly6G-FITC assay buffer was added and serum matrix was used. The (561105, RRID: AB_10562567), Ly6C-BV421 (562727, plate was read on a Luminex® 200™ instrument with RRID: AB_2737748), and I-A/I-E-BV771 (563414, RRID: xPONENT® software (Merck). AB_2738191), all from BD Biosciences, while the following lymphoid cell lineage antibodies were used: Clinical biochemistry analyses anti-mouse CD3-BV650 (564378, RRID: AB_2738779), Analyses for systemic toxicity assessment were per- CD4-BB700 (566407, RRID: AB_2744427), CD8-V500 formed by the Clinical Analysis Dept. Biochemistry at (560778, RRID: AB_1937329), CD25-PE (558642, Hospital Germans Trias i Pujol (HuGTiP), Badalona, RRID: AB_1645250), NK1⋅1-FITC (561082, RRID: Spain. Serum samples obtained at the endpoint of the AB_10563221), and FoxP3-Alexa647 (560401, RRID: experiment (n = 4 per treatment group, half of each AB_1645201), all from BD Biosciences. The cells for the gender) were analyzed by spectrophotometry using an lymphoid panel were then permeabilized and fixed AU5800 analyzer (Beckman Coulter, Passadena, CA, using the eBioscience™ FoxP3 staining buffer set USA). 6 www.thelancet.com Vol 91 May, 2023 Articles Quantification of IgG2a in mouse serum by ELISA RNA-sequencing (RNA-seq) and transcriptomic 5 μg/ml of capture Ab (anti-mouse IgG2a, 115-005-206, analysis Jackson ImmunoResearch, RRID:AB_2338462) were RNA-seq of CD45+CD11+Ly6C+Ly6Glo cells from mouse coated onto a microtiter plate overnight at 4 ◦ C. The tumors sorted by flow cytometry was performed by the plates were blocked for 1 h at RT with PBS containing Genome Analysis Platform of CIC bioGUNE (Bizkaia 5% BSA (A4503, Sigma–Aldrich). Next, 50 μL of mouse Science and Technology Park, Derio, Vizcaya). serum diluted 1:2000 in PBS-5% BSA was added and The quantity and quality of the isolated RNAs were incubated for 90 min at RT. Finally, 50 μL of HRP evaluated using the Qubit RNA HS Assay Kit (Q32855, conjugated anti-mouse IgG (A3673, Sigma–Aldrich, Thermo Fisher Scientific) and Agilent RNA 6000 Pico RRID: AB_258099) diluted 1:5000 in PBS −5% BSA Chips (5067-1513, Agilent Technologies, Santa Clara, was added for 1 h at RT. Color was developed as CA, USA), respectively. Sequencing libraries were pre- explained above. pared following the “TruSeq Stranded mRNA Sample Preparation Guide (Part 15031058 Rev. E)” using the RNA extraction and RT-qPCR “TruSeq® Stranded mRNA Library Prep” Kit RNA extraction from PB monocytes was performed as (20020594, Illumina, San Diego, CA, USA) and TruSeq previously described.17 Briefly, 5 × 105 cells/well RNA CD Index Plate (96 Indexes, 96 Samples) cultured in 24-well plates were incubated for 24 h in (20019792, Illumina). Raw reads were filtered using RPMI medium containing 5% FBS and the indicated skewer v0.2.233 to remove the low-quality reads and stimuli. Cells were then washed with PBS and disrupted trimming using the Illumina adapter. The STAR pro- with TRIzol reagent (15596026, Thermo Fisher Scien- gram34 against Mus musculus genome (GRCm38) was tific). Likewise, pellets from cells sorted by flow cytom- used to map the reads, followed by gene quantification etry (CD45+CD11+Ly6C+Ly6Glo) were also disrupted with the RSEM program35 using GENCODE m15 with TRIzol reagent. For RNA extraction from frozen reference annotation.36 After eliminating genes without mouse tumors, tissue was submerged in lysis buffer an expected value greater than 10, we used the quantile with β-mercaptoethanol and disrupted mechanically method and limma-voom transformation to normalize using a PRO 200® homogenizer. In all cases, total RNA non-biological variability. Differential expression was was extracted using the RNeasy Mini Kit (74106, Qia- assessed using moderated t-statistics.37 gen). The optional on-column DNase digestion using Principal Component Analysis from the expression the RNase-Free DNase set (79254, Qiagen, Hilden, matrix, heatmaps, and volcano plot were computed and Germany) was performed only on the samples that were plotted using R statistical software (v-4.2.0). The RNA- subsequently sequenced. RNA concentration was seq data are available from the GEO repository of the measured using a NanoDrop ND-1000 Spectrophotom- National Center for Biotechnology Information, U.S. eter (Thermo Fisher Scientific). All RNA samples were National Library of Medicine (the accession number for kept at −80 ◦ C until the qPCR was performed. Total these data is GEO: GSE208122). For automated func- RNA (0.5–1 μg) was reverse transcribed using the RNA tional annotation and classification of statistically sig- to cDNA EcoDry™ Premix (639549, Clontech, Moun- nificant genes based on GO Biological process terms, we tain View, CA, USA). Each RT reaction was then used DAVID.38 The protein interactome characterization amplified in a LightCycler® 480 PCR system using the of these genes was done with the Search Tool for the KAPA SYBR Fast Master Mix (KAPA Biosystems, Retrieval of Interacting Genes/Proteins (STRING) soft- 51230-100, Woburn, MA, USA). Samples were incu- ware (v11.5, ©String Consortium 2022). bated for an initial denaturation at 95 ◦ C for 5 min, then 40 PCR cycles were performed using the following Analysis of single-cell gene expression data from conditions: 95 ◦ C for 10 s, 60 ◦ C for 20 s, and 72 ◦ C for GEO127465 dataset 10 s. For CD5L, the RT reaction was amplified using the CD5L mRNA expression on lung cancer myeloid cell PrimeTime® Gene Expression Master Mix (1055772, population was analysed from available GEO dataset, by IDT, Newark, NJ, USA) under the following cycling working the GSE127465_human_counts_normali- program: polymerase activation at 95 ◦ C for 3 min, and zed_54773x41861.mtx.gz folder, integrated with 45 cycles of amplification consisting of denaturation for GSE127465_human_cell_metadata_54773x25.tsv.gz and 15 s at 95 ◦ C, and annealing/extension for 1 min at GSE127465_gene_names_human_41861.tsv.gz from 60 ◦ C. All primer pairs used in this study are listed in https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=G Supp Table S1. Gene expression values were normalized SE127465, by using python3 SCANPY.39 to the expression levels of the housekeeping genes GAPDH (glyceraldehyde 3-phosphate dehydrogenase) Statistical analysis for human samples and Actb for mouse samples. Fold Statistical analysis and Principal Component Analysis of induction levels were calculated using the average level the in vitro data were performed with GraphPad Prism of expression of each gene in control samples as a V.9.3.1 software (La Jolla, CA, USA). Specific statistical reference. tests are indicated in each figure legend. For survival www.thelancet.com Vol 91 May, 2023 7 Articles analysis, Kaplan–Meier’s method and the log-rank test induced CD5L protein expression (Fig. 1d, Supp Fig were performed to compare differences among curves. S6a). Therefore, our results reinforce the notion that Values of p ≤ 0.05 were considered significant. CD5L mRNA and protein expression is upregulated by tumor-derived stimuli. Role of funders The Funders had no role in study design, data collec- CD5L expression by TAMs correlates with poor tion, data analyses, interpretation, or writing of report. prognosis in human lung cancer We next investigated whether CD5L is expressed in TAMs in n = 55 specimens of human PAC, a subtype of Results NSCLC. The main clinical and pathological character- Cancer cells induce an immunosuppressive istics of the patients are summarized in Fig. 2a. The macrophage phenotype and stimulate CD5L IHC analysis revealed the presence of expression CD5L+ macrophages (Fig. 2b). Moreover, higher levels We sought to determine whether cancer cells from two of CD5L+ macrophages correlated with more advanced different types of human tumors, namely lung and liver stages of the disease (II–III) (median [range] of 6.3 gear macrophage activation towards immunosuppres- [2.0–8.4]) when compared to early (I) stages (4.0 sion and whether they influence CD5L expression. To [1.6–9.6]) (Fig. 2c). Furthermore, cases with higher TAM this end, we selected the A549 and PC9 cell lines, expression of CD5L showed a 3.5-fold increased likeli- derived from human Non-small-cell lung carcinoma hood of recurrence when compared to those with lower (NSCLC), as well as the human liver cancer cell lines expression (Fig. 2d). The co-expression of CD5L with Huh7, HepG2, and SNU398. CM of each different cell CD68, a pan-macrophage marker, was confirmed in six line were added to PB monocyte cultures, and changes cases by IF analysis (Fig. 2e) (Supp Fig S6b). Reinforc- in the expression of HLA-DR and macrophage activation ing these data, analysis of CD5L expression data ob- markers CD80, and CD23, CD163, and CD206 were tained from myeloid single cell transcriptomics in analyzed by flow cytometry. Alterations were compared human lung cancers (GSE127465) (Supp Fig S2a,b), to those induced by reference activation stimuli, namely suggest that CD5L mRNA is expressed in M2 and M0, IFN/LPS (proinflammatory), and IL4 and IL10 (two but not in M1 macrophages (Supp Fig S2c).40 Taken types of immunosuppressive cytokines).17 Collectively, together, these data indicate that infiltrating macro- the flow cytometry data were examined by Principal phages in lung cancer express CD5L, which in turn is Component Analysis (PCA) to reduce dimensionality. associated with a worse prognosis in patients with PCA analysis revealed that stimulation with cancer cell- NSCLC. CM induced a macrophage receptor expression pattern that predominantly resembled that of IL10-stimulated Anti-CD5L antibody RImAb inhibits IL10-induced macrophages (Fig. 1a). Interestingly, examination of activation the individual receptor expression showed that CM from To explore the therapeutic potential of targeting CD5L, lung A549 cells significantly increased the expression of we raised a function-blocking mAb against CD5L, which CD163. Likewise, lung PC9-CM increased the expres- we named RImAb. The antibody is an IgG2a kappa sion of CD23 and CD163 surface receptors (Supp Fig mAb, and, in a direct binding ELISA, it specifically in- S1). RT-qPCR analyses reinforced these findings, teracts with recombinant human CD5L (rhCD5L) but showing that treatment of PB monocytes with lung not with human DMBT1 (hDMBT1) or human CD5 cancer cell-CM increased the mRNA expression of (hCD5), two structurally related proteins with 44.64% CD163, CD206, VEGF and MERTK (Fig. 1b). All these and 36.36% sequence identity and 51.07% and 38.63% data collectively point to a significant effect of lung similarity, respectively, with CD5L in their SRCR do- cancer cell-CM on macrophages, driving them to an mains (Fig. 3a). Of note, RImAb cross-reacted with the immunosuppressive phenotype. Changes in receptor recombinant murine CD5L protein (rmCD5L) (69.29% expression by liver cancer cell-CM provided similar re- sequence identity and 73.97% similarity to its human sults, both by flow cytometry and RT-qPCR (Supp Fig S1 counterpart) (Fig. 3a), thus making it suitable for and Fig. 1b, respectively), thereby highlighting the experimental murine models. We then tested whether relevance of the results obtained in lung cancer for other RImAb would affect IL10-induced macrophage activa- types of solid cancers. tion. Interestingly, treatment with 5 μg/ml RImAb We next tested whether the CM altered CD5L before stimulation with IL10 increased the mRNA expression. Very low levels of CD5L mRNA were expression of the co-stimulatory molecule CD80 and the detected in non-stimulated macrophages, but these were pro-inflammatory cytokine TNFA (Fig. 3b), while significantly enhanced by all liver cancer-CM, as well as inhibiting the induction of the TAM-associated markers by lung cancer PC9-CM (Fig. 1c). Accordingly, IF CD163, VEGF, and, to a lesser extent, MERTK (Fig. 3b). staining of CD5L and confocal imaging showed that Accordingly, RImAb also inhibited the IL10-induced IL10, lung cancer PC9-CM and liver cancer HepG2-CM increase in CD5L mRNA expression (Fig. 3b), thereby 8 www.thelancet.com Vol 91 May, 2023 Articles a b c d Fig. 1: Lung and liver cancer cell-conditioned media (CM) induce an IL10-like phenotype and CD5L expression in macrophages. a) Principal Component Analysis (PCA) scatterplot of PB monocytes treated for 72 h with medium alone (control), reference activation stimuli www.thelancet.com Vol 91 May, 2023 9 Articles indicating that the IL10-induced phenotype can be pre- levels did not show any significant changes, we observed vented by CD5L blockade with RImAb. a significant increase in the iNOS/Arg-1 ratio (Mann– Whitney t-test; p = 0.0379) (Fig. 4d), suggesting a shift in Immunotherapy targeting CD5L (RImAb) inhibits TAMs towards the proinflammatory phenotype in the tumor growth, lowers the abundance of tumor RImAb-treated group. macrophages, and shifts the phenotype of this cell population RImAb treatment reprograms the intratumoral We next assessed CD5L as a target for immunotherapy myeloid cell compartment in a syngeneic mouse model of lung cancer. RImAb, We next used flow cytometry to study whether RImAb PBS, or the isotype control antibody was administered administration induced changes in tumor immune cell intraperitoneally (i.p.) every three days after tumor populations. The gating strategy for myeloid lineage is establishment (day 7). A schematic diagram summari- depicted in Fig. 5a. In these experiments, an imbalance zing the protocol is provided in Fig. 4a. RImAb treat- between the CD11b+Ly6ChiLy6Ghi (neutrophil popula- ment slowed tumor growth over time (Two-way tion) and CD11b+Ly6C+Ly6Glo (monocyte/macrophage ANOVA, p = 0.019). Consequently, at the end of the lineage) populations was observed, with a gain of the study, LLC tumor-bearing mice treated with RImAb former and loss of the latter in RImAb-treated animals presented statistically significant smaller tumors than (Fig. 5b and c, respectively). Within the monocyte/ those treated with PBS or the isotype control antibody macrophage populations, four subsets were delineated (Mann–Whitney t-test; p = 0.0093) (Fig. 4b). This on the basis of MHCII expression (Fig. 5d). Within experiment was performed four times with PBS (n = 2) Ly6Clow cells, considered TAMs,22,30,43 we observed an and IgG2a (n = 2) as control of the RImAb arm (n = 4), increase in MHCIIhi macrophages (Mann–Whitney t- with lower tumor size in RImAb-treated mice in all test; p = 0.0379), with no apparent changes in MHCIIlo cases (Supp Fig S3). ones (Mann–Whitney t-test, p = 0.2786) (Fig. 5d). Similar circulating levels of both RImAb and isotype Regarding Ly6Chi populations, considered infiltrating control antibodies were observed at the end of treatment monocytes,22,43 RImAb induced a decrease in those (Supp Fig S4a). Regarding potential toxicities, all the cells with MHCIIhi expression. In contrast, no changes animals showed comparable activity, alertness, and re- in the number of CD3+CD4+ or CD3+CD8+ T lym- action to stimuli. Their movement and posture were phocytes, Tregs (CD3+CD4+CD25+FoxP3+), nor NK normal. Their eyes were clear and showed no sign of T cells (CD3−NK.1.1+) were detected between treat- infection/inflammation. Neither did we observe any ment groups (see gating strategy in Fig. 5e) (Fig. 5f). To differences in weight, or in parameters reflecting liver further determine the potential differences in the function, namely alanine and aspartate transaminases lymphoid population, we analyzed the memory and (ALT and AST), renal function (creatinine), lipid meta- exhaustion state of intratumoral T-cells. Analysis of bolism, iron concentration, or bilirubin. Of note, all CCR7 and CD44 markers revealed no significant dif- these values were within the standard range, excluding ferences between the memory state of T-cells (Fig. 6a). potential functional organ/systemic impairment, as In contrast, analysis of exhaustion markers revealed described41,42 (Supp Fig S4b and c). that treatment with RImAb decreased the expression of We next used IF to examine macrophage abundance TIGIT in the CD4+ subpopulation (Fig. 6b), while no and phenotype in the tumor tissue and observed a sig- significant differences were observed in CTLA4+, nificant reduction in the number of F4/80+ macro- PD1+, LAG3+, and TIM3+ cells between treatment phages and of those expressing CD5L in the RImAb groups. Although a decrease in all exhaustion markers group compared to the controls (Mann–Whitney t-test; was observed in the CD8+ subpopulation, these were p = 0.0207 and p = 0.0110, respectively) (Fig. 4c, Supp not significant (Fig. 6b). Fig S6c). IHC using F4/80 confirmed the loss of F4/ Taken together, our data suggest that RImAb 80 staining, along with higher expression of iNOS administration affects mainly the proportion of intra- (Mann–Whitney t-test; p = 0.0281 and p = 0.054, tumoral myeloid cell lineages, although it also modu- respectively) (Fig. 4d). Even though Arginase-1 (Arg-1) lates the exhaustion state of CD4+ T-cells. (IFN/LPS, IL4, and IL10), or cancer cell-CM. Projection based on the expression profile of surface markers characterized by multicolor flow cytometry. PC1 and PC2 represented in each axis depict the first and second principal components, respectively. b) Expression of CD80, CD163, CD206, VEGF, MERTK and c) CD5L mRNA was assessed by RT-qPCR in PB monocytes treated for 24 h with the indicated stimuli. mRNA levels normalized to GAPDH, and fold induction levels were calculated using the average expression of each gene in control macrophages as a reference. Data are represented as mean ± SEM (n = 5 to 9). d) CD5L immunofluorescence staining (green) in macrophages treated with the indicated stimuli for 72 h. Nuclei were counterstained with Hoechst 33258 (blue). Scale bar represents 20 μm. CD5L mean fluorescence intensity (MFI) was calculated with ZenLite software and is represented as MFI ± SEM of 50 macrophages scored in random fields (right) (n = 3). Significance was calculated using the Mann–Whitney t-test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). 10 www.thelancet.com Vol 91 May, 2023 Articles a b c d e Fig. 2: CD5L expression by TAMs is associated with poor prognosis in papillary lung adenocarcinoma. a) Clinical characteristics of patients diagnosed with Papillary Adenocarcinoma (PAC) who participated in this study. b) Representative immunohistochemistry images showing CD5L expression in early (I) and advanced (II–III) stages of papillary lung adenocarcinoma. Scale bar represents 10 μm. c) Graph shows the number of CD5L+ macrophages per field in early (I, n = 35) and advanced (II–III, n = 20) stages. Data are presented as the mean ± SEM, *p < 0.01 determined by the Mann–Whitney t-test. d) Kaplan–Meier analysis of recurrence-free survival in cases with lower and higher TAM CD5L expression. The mean number of CD5L+ macrophages from stage I was taken as the limit value, *p < 0.01 determined by the Log-rank (Mantel– Cox) test. e) Representative immunofluorescence image depicting CD68+ (red) (i) and CD5L+ (green) macrophages, and their co-expression (orange), (ii). Scale bar represents 25 μm. RImAb administration induces profound of which 512 were downregulated, and 687 upregulated transcriptional changes in intratumoral monocyte/ (Fig. 7b). These were classified by biological process macrophage cells through DAVID bioinformatics software, which re- To further dissect how RImAb administration altered ported that they were significantly enriched in 15 bio- tumor populations of monocytes/macrophages, we sor- logical processes (p-val < 0.05) (Fig. 7c), of which our ted these cells (CD45+CD11b+Ly6C+Ly6Glo) and attention was drawn to angiogenesis, autophagy, innate analyzed their transcriptomic content by RNA-seq. immunity, and cell cycle processes because of their Sample-level quality control using PCA and clustering relevance in cancer. Variations in the expression of methods revealed that the replicates clustered within genes corresponding to these biological processes were two distinct groups associated with the distinct treat- then represented in a heatmap, which showed that ments (Fig. 7a). In this regard, we observed 1199 genes belonging to angiogenesis, autophagy, and innate differentially expressed genes (FC > ±1.5, p-val < 0.05), immunity processes were downregulated mainly in www.thelancet.com Vol 91 May, 2023 11 Articles a b Fig. 3: RImAb specifically binds to human and mouse CD5L and reverts the polarization induced by IL10. a) Direct ELISA of RImAb to hDMBT1, rhCD5, rmCD5L, rhCD5L, or BSA. A representative experiment of three performed is shown. b) RT-qPCR quantification of mRNA expression of CD80, TNFA, CD163, VEGF, MERTK, and CD5L in PB monocytes treated, when indicated, with 5 μg/ml of RImAb for 45 min before the addition of IL10 (50 ng/ml) for 24 h. mRNA levels relative to GAPDH, and fold induction levels were calculated using the expression of each gene in IL10-stimulated macrophages for each donor as a reference. Data are represented as mean ± SEM (n = 5 to 9). Significance was calculated using the Mann–Whitney t-test (*p ≤ 0.05). RImAb-treated mice (Fig. 7d). Regarding the genes addition, using STRING software, we represented the involved in the cell cycle, we observed a more variable interactome of up- and down-regulated proteins in pattern, with several genes upregulated and also down- RImAb-treated animals vs. the control group (Fig. 7e, regulated in RImAb-treated animals (Fig. 7d). In left and right, respectively). Several GO biological 12 www.thelancet.com Vol 91 May, 2023 Articles Fig. 4: Blockade of CD5L slows tumor growth in vivo and reprograms TAMs towards an antitumor profile. a) Study design and timeline for mouse model. b) LLC tumor growth in mm3 in mice treated with control (PBS) or the anti-CD5L RImAb. Data are presented as the mean ± SEM (n = 8 per group). *p < 0.01 determined by the two-way repeated measures ANOVA test. c) Left: Immunofluorescence demonstrating CD5L expression (red) by F4/80+ TAMs (green) in control (PBS) and RImAb-treated mice. Nuclei were counterstained with Hoechst 33258 (blue). Scale bars represent 25 μm. Right: graph representing the number of F4/80+ TAMs and F4/80+ TAMs expressing CD5L per field in control and RImAb- treated animals. Data are presented as the mean ± SEM (n = 8 per group). *p < 0.01 determined by the Mann–Whitney t-test. d) Immunohistochemistry depicting expression of F4/80, iNOS, and Arg-1 in tumor samples from control (PBS) and RImAb-treated mice. Scale www.thelancet.com Vol 91 May, 2023 13 Articles functions were identified as significantly enriched, the expression of Twist1 and Snail1, although the differ- most relevant related to DNA replication, repair and ences were not statistically significant (Mann–Whitney t- damage, and cell cycle for RImAb-upregulated genes, and test; p = 0.5358, and p = 0.4634, respectively) (Fig. 8e). to cytokine production, MAPK signaling, and angiogen- Importantly, RImAb-treated mice showed decreased esis for RImAb-downregulated genes (Fig. 7e, Supp tumor vascularity, as reflected by a significant reduction Table S2). Taken together, these results suggest that in the number of blood vessels compared to the controls RImAb administration significantly affects the tran- (Mann–Whitney t-test; p = 0.0140) when CD31 was used scriptomic profile of tumor monocyte/macrophages. (Fig. 8f). Moreover, the reduction in the number of blood vessels significantly correlated with the decrease RImAb administration leads to an enhanced in tumor volume (Mann–Whitney t-test; p = 0.0286) antitumor phenotype in the tumor (Supp Fig S5b). In line with these findings, the mRNA microenvironment expression of the potent angiogenic growth factor To achieve a deeper understanding of the changes in angiopoietin-2 (Angpt2) was greatly decreased in immune response caused by the treatment, we deter- RImAb-treated mice (Mann–Whitney t-test; p = 0.0023) mined additional proinflammatory markers by IHC. (Fig. 8g). These analyses showed increased myeloperoxidase (MPO) (Mann–Whitney t-test; p = 0.0281) and TNF-α (Mann–Whitney t-test; p = 0.0104) levels in the RImAb Discussion treated group (Fig. 8a). Interestingly, MPO expression Here we show that CD5L is an immune checkpoint in levels inversely correlated with tumor volume (Mann– macrophages. Importantly, CD5L protein overexpression Whitney t-test; p = 0.0442), (Supp Fig S5a). In addition, in TAMs correlated with a worse patient prognosis in lung we measured the intratumoral levels of key cytokines/ cancer. Moreover, through the action of a new specific chemokines. TNF-α expression was higher in RImAb- mAb, we demonstrate that targeting CD5L is a feasible treated mice, although not significant (Mann–Whitney approach to not only reprogram the macrophage tran- t-test; p = 0.4713). Moreover, these animals showed an scriptome and phenotype but also reduce the immune- increase in Granulocyte Macrophage Colony- suppressing characteristics of the TME, which together Stimulating Factor (GM-CSF), and in Macrophage In- result in reduced tumor growth in vivo. flammatory Protein-1 alpha (MIP-1α or CCL3) (Mann– Previously, we reported that IL10-activated macro- Whitney t-test; p = 0.0155). In contrast, RImAb-treated phages in vitro overexpressed CD5L.17 Through animals showed slightly lower expression of the che- comprehensive phenotypic profiling of human PB mokine RANTES ((C–C motif) ligand 5, CCL5) (Mann– monocytes using up to three complementary tech- Whitney t-test; p = 0.1374) (Fig. 8b). These same cyto- niques, we herein determined that CM from lung and kines were analyzed in serum, where we observed a liver cancer cells increase the expression of TAM significant decrease in GM-CSF in RImAb-treated ani- markers associated with immunosuppression while mals (Mann–Whitney t-test; p = 0.0071) (Supp Fig S4d). inducing CD5L levels. Interestingly, IL10, PC9-CM, and We then analyzed the expression of the immune HepG2-CM could increase the mRNA level of CD5L in checkpoint Ctla4 and Pdcd1, which were found to be macrophages, in which HepG2-CM exhibited a signifi- downregulated in the RImAb-treated vs. control mice cantly stronger stimulation of CD5L expression over (Mann–Whitney t-test; p = 0.0293 and p = 0.0350, PC9-CM. However, regarding the protein expression, respectively) (Fig. 8c). PC9-CM was associated with the highest MFI compared To broaden observations of the consequences of to IL10 and HepG2-CM. These apparently contradictory CD5L blockade, we next studied other markers of key results could be explained by previous studies suggest- aspects of tumorigenesis, namely cancer cell apoptosis, ing that CD5L expression is tightly regulated, and its the epithelial–mesenchymal transition (EMT), and tu- mRNA levels do not always correspond to those of its mor vascularity. RImAb administration led to a decrease protein.28,29,44–46 Additionally, in line with the previous in the apoptotic regulator BCL2, as analyzed by IHC results where CD5L induces an immunosuppressive (Mann–Whitney t-test; p = 0.0003) (Fig. 8d). In terms of phenotype,17 we determined that CD5L blockade rever- the EMT markers, a significant decrease in Zeb1 ted IL10-induced macrophage activation. Interestingly, (Mann–Whitney t-test; p = 0.041) expression was blocking CD5L also inhibited CD5L expression, sug- observed, concomitant with a tendency towards a lower gesting that this protein undergoes a positive feedback of bar represents 10 μm. Average stained area obtained from five random areas was calculated using Image J (color deconvolution) software. Graphs illustrate the average stained area for F4/80, iNOS, Arg-1 and ratio of iNOS/Arg-1 in control (PBS) and RImAb-treated mice. Data are presented as the mean ± SEM (n = 8 per group). *p < 0.01 determined by the Mann–Whitney t-test. 14 www.thelancet.com Vol 91 May, 2023 Articles a b SSC-A CD11b+Ly6ChiLy6Ghi FSC-A FSC-H CD11b 20 ✱ (% of CD45+cells) 15 Frequency FSC-A FSC-A CD45 F4/80 10 5 FSC-A Ly6C Ly6C 0 Control RImAb CD11b Ly6G MHCII c CD11b+Ly6C+Ly6Glo d Ly6CloMHCIIlo Ly6CloMHCIIhi Ly6ChiMHCIIlo Ly6ChiMHCIIhi ✱ ns 100 40 ns (% of CD45+CD11b+Ly6Glocells) 30 30 15 (% of CD45+CD11b+Ly6Glocells) (% of CD45+CD11b+Ly6Glocells) (% of CD45+CD11b+Ly6Glocells) ✱ ✱ 80 (% of CD45+cells) 30 Frequency Frequency 20 20 10 60 20 40 10 10 5 10 20 0 0 0 0 0 Control RImAb Control RImAb Control RImAb Isotype RImAb Isotype RImAb e SSC-A FSC-H NK1.1 FSC-H FSC-H FSC-A FSC-A CD45 CD3 CD3 FoxP3 CD25 CD8 CD8 CD25 CD4 CD4 CD3 f CD3+ CD4+ CD8+ CD3-NK1.1+ CD25+FoxP3+ 80 40 ns 1.5 30 ns 15 ns ns ns (% of CD45+CD3+CD4+cells) (% of CD45+CD3+cells) (% of CD45+CD3+Cells) (% of CD45+ cells) (% of CD45+cells) 60 30 10 1.0 20 Frequency 40 20 5 0.5 10 20 10 0 0 0 0.0 0 Control RImAb Control RImAb Control RImAb Control RImAb Control RImAb Fig. 5: RImAb modifies the intratumoral myeloid cell compartment in a mouse model of LLC. a) Myeloid cell gating classification using the expression of surface markers CD45, CD11b, Ly6C, Ly6G, and MHCII. b) Percentage of CD45+CD11b+Ly6ChiLy6Ghi neutrophil population. c) Percentage of CD45+CD11b+Ly6C+Ly6Glo myeloid cell subset. d) Percentage of CD45+CD11b+Ly6CloMHCIIlo, CD45+CD11b+Ly6CloMHCIIhi, CD45+CD11b+Ly6ChiMHCIIlo and CD45+CD11b+Ly6ChiMHCIIhi myeloid cell subsets. e) Lymphoid cell gating classification using the expression of the CD45, CD3, CD8, CD4, CD25, NK1.1, and FoxP3 markers. f) Percentage of each lymphocytic subset population analyzed in control (isotype IgG2a) and RImAb-treated mice. Data are presented as the mean of ±SEM (n = 8 per group), and statistical comparisons were performed using the Mann–Whitney t-test. www.thelancet.com Vol 91 May, 2023 15 Articles a b Fig. 6: RImAb modifies the exhaustion state of intratumoral T-cells in a mouse model of LLC. Flow cytometry analysis of a) CCR7 and CD44 memory markers, and b) CTLA4, PD1, LAG3, TIM3, TIGIT exhaustion markers, in CD4+ (upper panels) or CD8+ (lower panels) T-cells isolated from LLC tumor samples. Data are presented as the mean percentage with respect to CD3+ population ± SEM (n = 8 per group). *p < 0.01 determined by the Mann–Whitney t-test. transcriptional regulation. Together, these observations cells. Association of high TAM CD5L protein with thus point to a positive feedback loop between CD5L advanced stages of lung cancer disease and lower event- expression and the maintenance of the immunosuppres- free survival supports the notion that CD5L contributes sive phenotype.17 Therefore, we conclude that CD5L is a to a poor outcome in this pathology. It is also in accor- TAM marker that is induced by factors secreted by cancer dance with the presence of TAMs showing an inverse 16 www.thelancet.com Vol 91 May, 2023 Articles www.thelancet.com Vol 91 May, 2023 17 Articles correlation with prognosis and survival rates in this CD5L promotes autophagy,17,28 it is consistent that its pathology.47 blockage reduced the expression of autophagy genes. The generation of a specific antibody, namely Furthermore, the RImAb-treated group showed an RImAb, against CD5L allowed us to explore the thera- increase in the CD45+CD11b+Ly6ChiLy6Ghi population, peutic potential of targeting CD5L in the context of considered neutrophils,51 with respect to the controls. cancer. Of note, using PB monocytes in vitro, the addi- Neutrophils play important and conflicting roles in tion of RImAb before stimulation with IL10 reverted the cancer development.52,53 In our hands, the expression of phenotypic changes induced by this cytokine, thereby MPO, a marker of neutrophil activation, negatively reinforcing the notion that this mAb blocks macrophage correlated with tumor volume, thereby suggesting that immunosuppression. These data are in agreement with neutrophil infiltration contributes to tumor control by a significant increase in the iNOS/Arg-1 ratio in mice RImAb. The RImAb-associated increase in neutrophils treated with RImAb.48 is in line with the higher levels of tumor GM-CSF, as We sought to further characterize the monocyte/ this factor is a chemoattractant for neutrophils and may macrophage populations (CD45+CD11b+Ly6C+Ly6Glo) have antitumor activity.54,55 In contrast, we observed in tumor specimens from mice treated with RImAb. diminished levels of GM-CSF in the serum of RImAb- According to recent reports, these populations can be treated animals. Interestingly, increased GM-CSF differentiated into four subpopulations by flow cytom- serum levels are considered a marker of adverse clin- etry on the basis of Ly6C and MHCII expression, ical outcome, especially in patients with NSCLC.55 Ly6ChiMHCIIlo being inflammatory monocytes, RNAseq also revealed an imbalance in the regulation Ly6ChiMHCIIhi immature macrophages, Ly6CloMH- of cytokine production. Of the many factors involved in CIIhi TAMs, and Ly6CloMHCIIlo TAMs.22,43 Using this this process, we selected macrophage inflammatory categorization, we did not observe changes in protein-1 alpha (MIP-1α/CCL3), and RANTES as two Ly6CloMHCIIlo TAMs, which are described to be key macrophage-secreted chemokines.56,57 MIP-1α was immunosuppressive and tumor-promoting in vivo. increased in tumors from the RImAb group. This However, there was an increase in Ly6CloMHCIIhi observation is consistent with its described antitumoral TAMs, which have a higher antigen-presentation ca- role.57,58 On the other hand, RANTES levels were pacity and a more pro-inflammatory gene expression decreased in tumors from the RImAb-treated group. profile.7,30,43 These data reinforced the notion that This observation is relevant since this chemokine pro- RImAb administration alters the monocyte and macro- motes macrophage recruitment, immune evasion, and phage compartment within the tumor. In agreement, cancer cell invasion in different types of solid tumors, transcriptomic analyses of CD45+CD11b+Ly6C+Ly6Glo including NSCLC,58,59 and its gene expression is a pre- cells within the tumor revealed profound changes in dictor of survival in stage I NSCLC.60 Collectively, the mRNA expression between RImAb-treated and control changes in the expression of these two chemokines animals. Significant changes were observed in the indicate that RImAb enhances anti-tumoral immunity. expression of genes associated with the cell cycle, sug- The hallmarks of cancer proposed by Hanahan and gesting that the macrophage cell cycle is altered after Weinberg summarize the vast complexity of cancer treatment with RImAb. Likewise, genes involved in the mechanisms in a set of general principles.61,62 Accord- MAPK pathway were downregulated in RImAb-treated ingly, here we portray RImAb as a modulator of several mice, an observation that is consistent with the prolif- hallmarks, namely vascularity, apoptosis and epithelial- erative role of this pathway and its hyperactivation in to-mesenchymal (EMT) transition factors. RImAb cancer.49 Tightly connected to these two activities is the administration led to a notable decrease in tumor process of autophagy, whose associated genes were vascularity, as reflected by a reduction of VEGF in vitro, downregulated by RImAb. These data are consistent CD31 and Angpt2 in tissue, as well as differential with the participation of autophagy in regulating expression of Vegfa, Syk, and Kdr in tumor macrophages macrophage responses in cancer.50 Moreover, since in the RImAb-treated vs. control mice. Additionally, the Fig. 7: RImAb induces changes in the gene expression profile of the myeloid population in a mouse model of LLC. a) Principal Component Analysis (PCA) scatterplot of the gene expression profile of CD45+CD11b+Ly6C+Ly6Glo cells in control (isotype IgG2a) (n = 3) and RImAb (n = 4)- treated mice obtained by RNA-seq. Percentages represent variance captured by PCA 1 and 2. b) Volcano-plot representing genes differentially expressed in RImAb-treated vs. control (isotype IgG2a) group as detected by RNA-seq data sets. Red points mark the genes with significantly increased (right, 687 genes) or decreased (left, 512 genes) expression (p < 0.05). Gray line is set to p-value of 0.05. c) Gene Ontology (GO) enrichment analysis obtained from significantly differentially expressed genes (fold-change (FC) > 1.5, p-val < 0.05) between the control (isotype IgG2a) and RImAb-treated groups. Analysis was performed with DAVID database. d) Heatmaps of gene expression of the differentially expressed genes between the control (isotype IgG2a) and RImAb-treated groups for the GO Biological Processes of Angiogenesis, Autophagy, Innate Immunity, and Cell Cycle previously identified by DAVID. e) Interactome analysis of proteins coded by genes upregulated (left) and downregulated (right) in the RImAb-treated vs. control (isotype IgG2a) groups obtained with STRING (v11.5) software. 18 www.thelancet.com Vol 91 May, 2023 Articles a b c d e f g Fig. 8: RImAb reprograms the tumor microenvironment towards a more inflammatory and antitumoral phenotype in a mouse model of LLC. a) Left: Representative immunohistochemistry images of MPO and TNF-α expression in tumor samples from control (PBS) and RImAb- treated groups. Right: quantification of the average stained area of five random images obtained from each group. b) Levels of TNF-α, GM-CSF, MIP-1α, and RANTES cytokines in tumor homogenates, expressed as pg cytokine/mg protein, in control (isotype IgG2a) and RImAb-treated mice (8 mice per group), measured by the Multiplex Assay. Bar graphs show means with SEM. c) Expression of Ctla4 and Pdcd1 mRNA was assessed by RT-qPCR in total tumor from control (isotype IgG2a) and RImAb-treated mice. d) Left: Representative immunohistochemistry images of BCL2 expression in tumor samples from the control (PBS) and RImAb-treated mice. Right: quantification of the average stained area of 5 random images obtained from each group. e) Expression of EMT markers Zeb1, Snail1, and Twist1 mRNA was assessed by RT-qPCR in total www.thelancet.com Vol 91 May, 2023 19 Articles RImAb-treated mice showed a significant reduction of Founder and CEO of AlbaJuna Therapeutics S.L. from which he owns BCL2, thereby suggesting a decrease in anti-apoptotic stock options. He is also consultant for MSD and Nesapor S.L, and received support from Gilead for attending meetings. GT has received activity, which may lead to increased apoptosis and honoraria from Takeda for lectures. effective tumor killing. Treatment with RImAb also decreased the expression of Zeb1 but not Snai1 or Acknowledgements Twist1. Strikingly, it has been reported that TAMs We thank Gerard Requena (Flow Cytometry Platform, IGTP) and M. require ZEB1 for their tumor-promoting functions, and Pilar Armengol (Translational Genomics Platform and Microscopy only TAMs that express full levels of ZEB1 accelerate Platform, IGTP) for their valuable support in flow cytometry studies, and RNA and microscopy analysis, respectively. We also wish to tumor growth.63 acknowledge Maria Doladé Botías (Clinical Analyses and Biochem- The present study reveals that reprogramming istry Dept., HUGTiP) for analyzing the mouse serum samples; Dr. Jo macrophages in the TME using an anti-CD5L antibody van Ginderachter (VIB, Belgium) for providing 3LL-R cells; Ramon is safe in mice. Given this finding, this approach may Bartolí (IGTP-CIBERehd) for help writing the CMCIB ethical proto- have potential applications for immunotherapy in lung col for Animal Experimentation; Ana M Aransay for performing the RNAseq experiments (Genome Analysis Platform, CIC bioGUNE, cancer and also in other malignancies.24 Moreover, it Spain); Imane Chaib (Cancer Biology and Precision Medicine labo- would be interesting to study whether RImAb enhances ratory of Dr. Rosell ICO-IGTP) for providing the A459 and PC9 lung the effect of T-cell-directed immunotherapies, such as cancer cell lines used in the study; Elena Ramos for help with anti- those that target the immune checkpoints TIGIT, body purification (Dept. of Immunology and Oncology, CNB-CSIC) and Ismael Varela for his technical assistance with in vivo mice CTLA4 and PD-L1, as these were found to be down- experiments. regulated in CD4+ T-cells and whole tumors from This study was supported by the Instituto de Salud Carlos III RImAb-treated mice, respectively. (ISCIII), and ERDFs from the EU, ‘Una manera de hacer Europa’, In conclusion, here we reveal a key function for grants PI19/00523, 2019 PROD 00118 AGAUR, CI20-00152 and CC22- CD5L protein in modulating the activity of macrophages 10181 Caixa Impulse to MRS, and from the Spanish Ministry of Science and Innovation grant PID2020-119875RB-I00 and PID2020-105404RB- and their interactions within the TME. We further I00 to AFV and LK, respectively. This research was also supported by the propose a new approach against the immunosuppres- Consorcio Centro de Investigación Biomédica en Red (CIBERES and sive TME based on targeting CD5L with a new mAb, CIBEREHD). LSM, TP, JFD, and CM are supported by Juan de la Cierva which emerges as a promising approach to cancer (FJC2019-041213-I), PFIS (FI20/00115), FPI (PRE2018-085579), and Margarita Salas (MSGD2021-08) fellowships, respectively, all of which immunotherapy. are programs run by the Spanish Government and NextGenerationEU, respectively. This study received funding from the EU’s Horizon 2020 Contributors Research and Development Program under grant agreement nº 847762. LSM, TP, CM, JFD, LS, GA, ET, JC, AFV, LK, and MRS: designed and The Innate Immunity lab and the UTE are accredited by the Catalan performed research, analysed data; MI: contributed patient samples and Agency for Management of University and Research Grants 2017-SGR- clinical databases; JB: designed research; MT, HD: performed research; 490/2021-SGR-01186 and 2017-SGR-500/2021 SGR 00920, respectively. GT, CV: designed and performed research and analysed data; MAFS: MRS, CV, and CA are researchers at IGTP, which is a member of the designed and analysed data; CF, JS, HM, JJL, ARC, and CA: analysed CERCA network of institutes supported by the Health Department of data; LSM, TP, CM, and MRS wrote the paper. All authors read and the Government of Catalonia. approved the final version of the manuscript. LSM, TP, CM, and MRS have verified the underlying data. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi. Data sharing statement org/10.1016/j.ebiom.2023.104555. Data reported in this paper will be shared by the corresponding author upon request. This study generated datasets available from the GEO with accession number GEO: GSE208122. References Declaration of interests 1 Whiteside TL. The tumor microenvironment and its role in pro- moting tumor growth. Oncogene. 2008;27(45):5904–5912. A patent protecting a method for the detection of CD5L has been sub- 2 Bejarano L, Jordāo MJ, Joyce JA, Bejarano L, Jordāo M, Author C. mitted to the European Patent Office (EP3653646A1). Likewise, the Therapeutic targeting of the tumor microenvironment. Cancer RImAb antibody is the object of an EP3476863A1 patent. LK is part of an Discov. 2021;11(4):933–959. institutional licensing agreement with SunRock Biopharma, and co- 3 Cassetta L, Pollard JW. 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EMBO J. 2017;36(22):3336. 22 www.thelancet.com Vol 91 May, 2023