Immunopeptidomics-based identification of naturally presented non-canonical circRNA-derived peptides.

Circular RNAs (circRNAs) are covalently closed non-coding RNAs lacking the 5' cap and the poly-A tail. Nevertheless, it has been demonstrated that certain circRNAs can undergo active translation. Therefore, aberrantly expressed circRNAs in human cancers could be an unexplored source of tumor-specific antigens, potentially mediating anti-tumor T cell responses. This study presents an immunopeptidomics workflow with a specific focus on generating a circRNA-specific protein fasta reference. The main goal of this workflow is to streamline the process of identifying and validating human leukocyte antigen (HLA) bound peptides potentially originating from circRNAs. We increase the analytical stringency of our workflow by retaining peptides identified independently by two mass spectrometry search engines and/or by applying a group-specific FDR for canonical-derived and circRNA-derived peptides. A subset of circRNA-derived peptides specifically encoded by the region spanning the back-splice junction (BSJ) are validated with targeted MS, and with direct Sanger sequencing of the respective source transcripts. Our workflow identifies 54 unique BSJ-spanning circRNA-derived peptides in the immunopeptidome of melanoma and lung cancer samples. Our approach enlarges the catalog of source proteins that can be explored for immunotherapy.

Machine learning methods and harmonized datasets improve immunogenic neoantigen prediction.

The accurate selection of neoantigens that bind to class I human leukocyte antigen (HLA) and are recognized by autologous T cells is a crucial step in many cancer immunotherapy pipelines. We reprocessed whole-exome sequencing and RNA sequencing (RNA-seq) data from 120 cancer patients from two external large-scale neoantigen immunogenicity screening assays combined with an in-house dataset of 11 patients and identified 46,017 somatic single-nucleotide variant mutations and 1,781,445 neo-peptides, of which 212 mutations and 178 neo-peptides were immunogenic. Beyond features commonly used for neoantigen prioritization, factors such as the location of neo-peptides within protein HLA presentation hotspots, binding promiscuity, and the role of the mutated gene in oncogenicity were predictive for immunogenicity. The classifiers accurately predicted neoantigen immunogenicity across datasets and improved their ranking by up to 30%. Besides insights into machine learning methods for neoantigen ranking, we have provided homogenized datasets valuable for developing and benchmarking companion algorithms for neoantigen-based immunotherapies.

A phase 1 trial of adoptive transfer of vaccine-primed autologous circulating T cells in ovarian cancer.

We have previously shown that vaccination with tumor-pulsed dendritic cells amplifies neoantigen recognition in ovarian cancer. Here, in a phase 1 clinical study ( NCT01312376 /UPCC26810) including 19 patients, we show that such responses are further reinvigorated by subsequent adoptive transfer of vaccine-primed, ex vivo-expanded autologous peripheral blood T cells. The treatment is safe, and epitope spreading with novel neopeptide reactivities was observed after cell infusion in patients who experienced clinical benefit, suggesting reinvigoration of tumor-sculpting immunity.

Neoantigen-specific CD8 T cells with high structural avidity preferentially reside in and eliminate tumors.

The success of cancer immunotherapy depends in part on the strength of antigen recognition by T cells. Here, we characterize the T cell receptor (TCR) functional (antigen sensitivity) and structural (monomeric pMHC-TCR off-rates) avidities of 371 CD8 T cell clones specific for neoantigens, tumor-associated antigens (TAAs) or viral antigens isolated from tumors or blood of patients and healthy donors. T cells from tumors exhibit stronger functional and structural avidity than their blood counterparts. Relative to TAA, neoantigen-specific T cells are of higher structural avidity and, consistently, are preferentially detected in tumors. Effective tumor infiltration in mice models is associated with high structural avidity and CXCR3 expression. Based on TCR biophysicochemical properties, we derive and apply an in silico model predicting TCR structural avidity and validate the enrichment in high avidity T cells in patients' tumors. These observations indicate a direct relationship between neoantigen recognition, T cell functionality and tumor infiltration. These results delineate a rational approach to identify potent T cells for personalized cancer immunotherapy.

The immunopeptidome landscape associated with T cell infiltration, inflammation and immune editing in lung cancer.

One key barrier to improving efficacy of personalized cancer immunotherapies that are dependent on the tumor antigenic landscape remains patient stratification. Although patients with CD3+CD8+ T cell-inflamed tumors typically show better response to immune checkpoint inhibitors, it is still unknown whether the immunopeptidome repertoire presented in highly inflamed and noninflamed tumors is substantially different. We surveyed 61 tumor regions and adjacent nonmalignant lung tissues from 8 patients with lung cancer and performed deep antigen discovery combining immunopeptidomics, genomics, bulk and spatial transcriptomics, and explored the heterogeneous expression and presentation of tumor (neo)antigens. In the present study, we associated diverse immune cell populations with the immunopeptidome and found a relatively higher frequency of predicted neoantigens located within HLA-I presentation hotspots in CD3+CD8+ T cell-excluded tumors. We associated such neoantigens with immune recognition, supporting their involvement in immune editing. This could have implications for the choice of combination therapies tailored to the patient's mutanome and immune microenvironment.

Cancer vaccines based on whole-tumor lysate or neoepitopes with validated HLA binding outperform those with predicted HLA-binding affinity.

Antigen selection and prioritization represent crucial determinants of vaccines' efficacy. Here, we compare two personalized dendritic cell-based vaccination strategies using whole-tumor lysate or neoantigens. Data in mouse and in cancer patients demonstrate that peptide vaccines using neoantigens predicted on the sole basis of in silico peptide-major histocompatibility complex (MHC) binding affinity underperform relative to whole-tumor-lysate vaccines. In contrast, effective in vitro peptide-MHC binding affinity and peptide immunogenicity significantly improve the prioritization of tumor-rejecting neoepitopes and result in more efficacious vaccines.

Bedside formulation of a personalized multi-neoantigen vaccine against mammary carcinoma.

BACKGROUND: Harnessing the immune system to purposely recognize and destroy tumors represents a significant breakthrough in clinical oncology. Non-synonymous mutations (neoantigenic peptides) were identified as powerful cancer targets. This knowledge can be exploited for further improvements of active immunotherapies, including cancer vaccines, as T cells specific for neoantigens are not attenuated by immune tolerance mechanism and do not harm healthy tissues. The current study aimed at developing an optimized multitarget vaccine using short or long neoantigenic peptides utilizing virus-like particles (VLPs) as an efficient vaccine platform. METHODS: Mutations of murine mammary carcinoma cells were identified by integrating mass spectrometry-based immunopeptidomics and whole exome sequencing. Neoantigenic peptides were synthesized and covalently linked to virus-like nanoparticles using a Cu-free click chemistry method for easy preparation of vaccines against mouse mammary carcinoma. RESULTS: As compared with short peptides, vaccination with long peptides was superior in the generation of neoantigen-specific CD4+ and CD8+ T cells, which readily produced interferon gamma (IFN-γ) and tumor-necrosis factor α (TNF-α). The resulting anti-tumor effect was associated with favorable immune re-polarization in the tumor microenvironment through reduction of myeloid-derived suppressor cells. Vaccination with long neoantigenic peptides also decreased post-surgical tumor recurrence and metastases, and prolonged mouse survival, despite the tumor's low mutational burden. CONCLUSION: Integrating mass spectrometry-based immunopeptidomics and whole exome sequencing is an efficient approach for identifying neoantigenic peptides. Our multitarget VLP-based vaccine shows a promising anti-tumor effect in an aggressive murine mammary carcinoma model. Future clinical application using this strategy is readily feasible and practical, as click chemistry coupling of personalized synthetic peptides to the nanoparticles can be done at the bedside directly before injection.

Sensitive identification of neoantigens and cognate TCRs in human solid tumors.

The identification of patient-specific tumor antigens is complicated by the low frequency of T cells specific for each tumor antigen. Here we describe NeoScreen, a method that enables the sensitive identification of rare tumor (neo)antigens and of cognate T cell receptors (TCRs) expressed by tumor-infiltrating lymphocytes. T cells transduced with tumor antigen-specific TCRs identified by NeoScreen mediate regression of established tumors in patient-derived xenograft mice.

Cell-autonomous inflammation of BRCA1-deficient ovarian cancers drives both tumor-intrinsic immunoreactivity and immune resistance via STING.

In this study, we investigate mechanisms leading to inflammation and immunoreactivity in ovarian tumors with homologous recombination deficiency (HRD). BRCA1 loss is found to lead to transcriptional reprogramming in tumor cells and cell-intrinsic inflammation involving type I interferon (IFN) and stimulator of IFN genes (STING). BRCA1-mutated (BRCA1mut) tumors are thus T cell inflamed at baseline. Genetic deletion or methylation of DNA-sensing/IFN genes or CCL5 chemokine is identified as a potential mechanism to attenuate T cell inflammation. Alternatively, in BRCA1mut cancers retaining inflammation, STING upregulates VEGF-A, mediating immune resistance and tumor progression. Tumor-intrinsic STING elimination reduces neoangiogenesis, increases CD8+ T cell infiltration, and reverts therapeutic resistance to dual immune checkpoint blockade (ICB). VEGF-A blockade phenocopies genetic STING loss and synergizes with ICB and/or poly(ADP-ribose) polymerase (PARP) inhibitors to control the outgrowth of Trp53-/-Brca1-/- but not Brca1+/+ ovarian tumors in vivo, offering rational combinatorial therapies for HRD cancers.

Sensitive Immunopeptidomics by Leveraging Available Large-Scale Multi-HLA Spectral Libraries, Data-Independent Acquisition, and MS/MS Prediction.

Mass spectrometry (MS) is the state-of-the-art methodology for capturing the breadth and depth of the immunopeptidome across human leukocyte antigen (HLA) allotypes and cell types. The majority of studies in the immunopeptidomics field are discovery driven. Hence, data-dependent tandem MS (MS/MS) acquisition (DDA) is widely used, as it generates high-quality references of peptide fingerprints. However, DDA suffers from the stochastic selection of abundant ions that impairs sensitivity and reproducibility. In contrast, in data-independent acquisition (DIA), the systematic fragmentation and acquisition of all fragment ions within given isolation m/z windows yield a comprehensive map for a given sample. However, many DIA approaches commonly require generating comprehensive DDA-based spectrum libraries, which can become impractical for studying noncanonical and personalized neoantigens. Because the amount of HLA peptides eluted from biological samples such as small tissue biopsies is typically not sufficient for acquiring both meaningful DDA data necessary for generating comprehensive spectral libraries and DIA MS measurements, the implementation of DIA in the immunopeptidomics translational research domain has remained limited. We implemented a DIA immunopeptidomics workflow and assessed its sensitivity and accuracy by matching DIA data against libraries with growing complexity-from sample-specific libraries to libraries combining 2 to 40 different immunopeptidomics samples. Analyzing DIA immunopeptidomics data against a complex multi-HLA spectral library resulted in a two-fold increase in peptide identification compared with sample-specific library and in a three-fold increase compared with DDA measurements, yet with no detrimental effect on the specificity. Furthermore, we demonstrated the implementation of DIA for sensitive personalized neoantigen discovery through the analysis of DIA data with predicted MS/MS spectra of clinically relevant HLA ligands. We conclude that a comprehensive multi-HLA library for DIA approach in combination with MS/MS prediction is highly advantageous for clinical immunopeptidomics, especially when low amounts of biological samples are available.

Personalized cancer vaccine strategy elicits polyfunctional T cells and demonstrates clinical benefits in ovarian cancer.

T cells are important for controlling ovarian cancer (OC). We previously demonstrated that combinatorial use of a personalized whole-tumor lysate-pulsed dendritic cell vaccine (OCDC), bevacizumab (Bev), and cyclophosphamide (Cy) elicited neoantigen-specific T cells and prolonged OC survival. Here, we hypothesize that adding acetylsalicylic acid (ASA) and low-dose interleukin (IL)-2 would increase the vaccine efficacy in a recurrent advanced OC phase I trial (NCT01132014). By adding ASA and low-dose IL-2 to the OCDC-Bev-Cy combinatorial regimen, we elicited vaccine-specific T-cell responses that positively correlated with patients' prolonged time-to-progression and overall survival. In the ID8 ovarian model, animals receiving the same regimen showed prolonged survival, increased tumor-infiltrating perforin-producing T cells, increased neoantigen-specific CD8+ T cells, and reduced endothelial Fas ligand expression and tumor-infiltrating T-regulatory cells. This combinatorial strategy was efficacious and also highlighted the predictive value of the ID8 model for future ovarian trial development.

CIITA-Transduced Glioblastoma Cells Uncover a Rich Repertoire of Clinically Relevant Tumor-Associated HLA-II Antigens.

CD4+ T cell responses are crucial for inducing and maintaining effective anticancer immunity, and the identification of human leukocyte antigen class II (HLA-II) cancer-specific epitopes is key to the development of potent cancer immunotherapies. In many tumor types, and especially in glioblastoma (GBM), HLA-II complexes are hardly ever naturally expressed. Hence, little is known about immunogenic HLA-II epitopes in GBM. With stable expression of the class II major histocompatibility complex transactivator (CIITA) coupled to a detailed and sensitive mass spectrometry-based immunopeptidomics analysis, we here uncovered a remarkable breadth of the HLA-ligandome in HROG02, HROG17, and RA GBM cell lines. The effect of CIITA expression on the induction of the HLA-II presentation machinery was striking in each of the three cell lines, and it was significantly higher compared with interferon gamma (IFNÉ£) treatment. In total, we identified 16,123 unique HLA-I peptides and 32,690 unique HLA-II peptides. In order to genuinely define the identified peptides as true HLA ligands, we carefully characterized their association with the different HLA allotypes. In addition, we identified 138 and 279 HLA-I and HLA-II ligands, respectively, most of which are novel in GBM, derived from known GBM-associated tumor antigens that have been used as source proteins for a variety of GBM vaccines. Our data further indicate that CIITA-expressing GBM cells acquired an antigen presenting cell-like phenotype as we found that they directly present external proteins as HLA-II ligands. Not only that CIITA-expressing GBM cells are attractive models for antigen discovery endeavors, but also such engineered cells have great therapeutic potential through massive presentation of a diverse antigenic repertoire.

Integrated proteogenomic deep sequencing and analytics accurately identify non-canonical peptides in tumor immunopeptidomes.

Efforts to precisely identify tumor human leukocyte antigen (HLA) bound peptides capable of mediating T cell-based tumor rejection still face important challenges. Recent studies suggest that non-canonical tumor-specific HLA peptides derived from annotated non-coding regions could elicit anti-tumor immune responses. However, sensitive and accurate mass spectrometry (MS)-based proteogenomics approaches are required to robustly identify these non-canonical peptides. We present an MS-based analytical approach that characterizes the non-canonical tumor HLA peptide repertoire, by incorporating whole exome sequencing, bulk and single-cell transcriptomics, ribosome profiling, and two MS/MS search tools in combination. This approach results in the accurate identification of hundreds of shared and tumor-specific non-canonical HLA peptides, including an immunogenic peptide derived from an open reading frame downstream of the melanoma stem cell marker gene ABCB5. These findings hold great promise for the discovery of previously unknown tumor antigens for cancer immunotherapy.

A Phase I/II trial comparing autologous dendritic cell vaccine pulsed either with personalized peptides (PEP-DC) or with tumor lysate (OC-DC) in patients with advanced high-grade ovarian serous carcinoma.

BACKGROUND: Most ovarian cancer patients are diagnosed at a late stage with 85% of them relapsing after surgery and standard chemotherapy; for this reason, new treatments are urgently needed. Ovarian cancer has become a candidate for immunotherapy by reason of their expression of shared tumor-associated antigens (TAAs) and private mutated neoantigens (NeoAgs) and the recognition of the tumor by the immune system. Additionally, the presence of intraepithelial tumor infiltrating lymphocytes (TILs) is associated with improved progression-free and overall survival of patients with ovarian cancer. The aim of active immunotherapy, including vaccination, is to generate a new anti-tumor response and amplify an existing immune response. Recently developed NeoAgs-based cancer vaccines have the advantage of being more tumor specific, reducing the potential for immunological tolerance, and inducing robust immunogenicity. METHODS: We propose a randomized phase I/II study in patients with advanced ovarian cancer to compare the immunogenicity and to assess safety and feasibility of two personalized DC vaccines. After standard of care surgery and chemotherapy, patients will receive either a novel vaccine consisting of autologous DCs pulsed with up to ten peptides (PEP-DC), selected using an agnostic, yet personalized, epitope discovery algorithm, or a sequential combination of a DC vaccine loaded with autologous oxidized tumor lysate (OC-DC) prior to an equivalent PEP-DC vaccine. All vaccines will be administered in combination with low-dose cyclophosphamide. This study is the first attempt to compare the two approaches and to use NeoAgs-based vaccines in ovarian cancer in the adjuvant setting. DISCUSSION: The proposed treatment takes advantage of the beneficial effects of pre-treatment with OC-DC prior to PEP-DC vaccination, prompting immune response induction against a wide range of patient-specific antigens, and amplification of pre-existing NeoAgs-specific T cell clones. Trial registration This trial is already approved by Swissmedic (Ref.: 2019TpP1004) and will be registered at http://www.clinicaltrials.gov before enrollment opens.

Immunopeptidomics of colorectal cancer organoids reveals a sparse HLA class I neoantigen landscape and no increase in neoantigens with interferon or MEK-inhibitor treatment.

BACKGROUND: Patient derived organoids (PDOs) can be established from colorectal cancers (CRCs) as in vitro models to interrogate cancer biology and its clinical relevance. We applied mass spectrometry (MS) immunopeptidomics to investigate neoantigen presentation and whether this can be augmented through interferon gamma (IFNγ) or MEK-inhibitor treatment. METHODS: Four microsatellite stable PDOs from chemotherapy refractory and one from a treatment naïve CRC were expanded to replicates with 100 million cells each, and HLA class I and class II peptide ligands were analyzed by MS. RESULTS: We identified an average of 9936 unique peptides per PDO which compares favorably against published immunopeptidomics studies, suggesting high sensitivity. Loss of heterozygosity of the HLA locus was associated with low peptide diversity in one PDO. Peptides from genes without detectable expression by RNA-sequencing were rarely identified by MS. Only 3 out of 612 non-silent mutations encoded for neoantigens that were detected by MS. In contrast, computational HLA binding prediction estimated that 304 mutations could generate neoantigens. One hundred ninety-six of these were located in expressed genes, still exceeding the number of MS-detected neoantigens 65-fold. Treatment of four PDOs with IFNγ upregulated HLA class I expression and qualitatively changed the immunopeptidome, with increased presentation of IFNγ-inducible genes. HLA class II presented peptides increased dramatically with IFNγ treatment. MEK-inhibitor treatment showed no consistent effect on HLA class I or II expression or the peptidome. Importantly, no additional HLA class I or II presented neoantigens became detectable with any treatment. CONCLUSIONS: Only 3 out of 612 non-silent mutations encoded for neoantigens that were detectable by MS. Although MS has sensitivity limits and biases, and likely underestimated the true neoantigen burden, this established a lower bound of the percentage of non-silent mutations that encode for presented neoantigens, which may be as low as 0.5%. This could be a reason for the poor responses of non-hypermutated CRCs to immune checkpoint inhibitors. MEK-inhibitors recently failed to improve checkpoint-inhibitor efficacy in CRC and the observed lack of HLA upregulation or improved peptide presentation may explain this.

A Phase Ib Study of the Combination of Personalized Autologous Dendritic Cell Vaccine, Aspirin, and Standard of Care Adjuvant Chemotherapy Followed by Nivolumab for Resected Pancreatic Adenocarcinoma-A Proof of Antigen Discovery Feasibility in Three Patients.

Despite the promising therapeutic effects of immune checkpoint blockade (ICB), most patients with solid tumors treated with anti-PD-1/PD-L1 monotherapy do not achieve objective responses, with most tumor regressions being partial rather than complete. It is hypothesized that the absence of pre-existing antitumor immunity and/or the presence of additional tumor immune suppressive factors at the tumor microenvironment are responsible for such therapeutic failures. It is therefore clear that in order to fully exploit the potential of PD-1 blockade therapy, antitumor immune response should be amplified, while tumor immune suppression should be further attenuated. Cancer vaccines may prime patients for treatments with ICB by inducing effective anti-tumor immunity, especially in patients lacking tumor-infiltrating T-cells. These "non-inflamed" non-permissive tumors that are resistant to ICB could be rendered sensitive and transformed into "inflamed" tumor by vaccination. In this article we describe a clinical study where we use pancreatic cancer as a model, and we hypothesize that effective vaccination in pancreatic cancer patients, along with interventions that can reprogram important immunosuppressive factors in the tumor microenvironment, can enhance tumor immune recognition, thus enhancing response to PD-1/PD-L1 blockade. We incorporate into the schedule of standard of care (SOC) chemotherapy adjuvant setting a vaccine platform comprised of autologous dendritic cells loaded with personalized neoantigen peptides (PEP-DC) identified through our own proteo-genomics antigen discovery pipeline. Furthermore, we add nivolumab, an antibody against PD-1, to boost and maintain the vaccine's effect. We also demonstrate the feasibility of identifying personalized neoantigens in three pancreatic ductal adenocarcinoma (PDAC) patients, and we describe their optimal incorporation into long peptides for manufacturing into vaccine products. We finally discuss the advantages as well as the scientific and logistic challenges of such an exploratory vaccine clinical trial, and we highlight its novelty.

Personalized cancer vaccine effectively mobilizes antitumor T cell immunity in ovarian cancer.

We conducted a pilot clinical trial testing a personalized vaccine generated by autologous dendritic cells (DCs) pulsed with oxidized autologous whole-tumor cell lysate (OCDC), which was injected intranodally in platinum-treated, immunotherapy-naïve, recurrent ovarian cancer patients. OCDC was administered alone (cohort 1, n = 5), in combination with bevacizumab (cohort 2, n = 10), or bevacizumab plus low-dose intravenous cyclophosphamide (cohort 3, n = 10) until disease progression or vaccine exhaustion. A total of 392 vaccine doses were administered without serious adverse events. Vaccination induced T cell responses to autologous tumor antigen, which were associated with significantly prolonged survival. Vaccination also amplified T cell responses against mutated neoepitopes derived from nonsynonymous somatic tumor mutations, and this included priming of T cells against previously unrecognized neoepitopes, as well as novel T cell clones of markedly higher avidity against previously recognized neoepitopes. We conclude that the use of oxidized whole-tumor lysate DC vaccine is safe and effective in eliciting a broad antitumor immunity, including private neoantigens, and warrants further clinical testing.

Sensitive and frequent identification of high avidity neo-epitope specific CD8 + T cells in immunotherapy-naive ovarian cancer.

Immunotherapy directed against private tumor neo-antigens derived from non-synonymous somatic mutations is a promising strategy of personalized cancer immunotherapy. However, feasibility in low mutational load tumor types remains unknown. Comprehensive and deep analysis of circulating and tumor-infiltrating lymphocytes (TILs) for neo-epitope specific CD8+ T cells has allowed prompt identification of oligoclonal and polyfunctional such cells from most immunotherapy-naive patients with advanced epithelial ovarian cancer studied. Neo-epitope recognition is discordant between circulating T cells and TILs, and is more likely to be found among TILs, which display higher functional avidity and unique TCRs with higher predicted affinity than their blood counterparts. Our results imply that identification of neo-epitope specific CD8+ T cells is achievable even in tumors with relatively low number of somatic mutations, and neo-epitope validation in TILs extends opportunities for mutanome-based personalized immunotherapies to such tumors.

Cortical-Bone Fragility--Insights from sFRP4 Deficiency in Pyle's Disease.

BACKGROUND: Cortical-bone fragility is a common feature in osteoporosis that is linked to nonvertebral fractures. Regulation of cortical-bone homeostasis has proved elusive. The study of genetic disorders of the skeleton can yield insights that fuel experimental therapeutic approaches to the treatment of rare disorders and common skeletal ailments. METHODS: We evaluated four patients with Pyle's disease, a genetic disorder that is characterized by cortical-bone thinning, limb deformity, and fractures; two patients were examined by means of exome sequencing, and two were examined by means of Sanger sequencing. After a candidate gene was identified, we generated a knockout mouse model that manifested the phenotype and studied the mechanisms responsible for altered bone architecture. RESULTS: In all affected patients, we found biallelic truncating mutations in SFRP4, the gene encoding secreted frizzled-related protein 4, a soluble Wnt inhibitor. Mice deficient in Sfrp4, like persons with Pyle's disease, have increased amounts of trabecular bone and unusually thin cortical bone, as a result of differential regulation of Wnt and bone morphogenetic protein (BMP) signaling in these two bone compartments. Treatment of Sfrp4-deficient mice with a soluble Bmp2 receptor (RAP-661) or with antibodies to sclerostin corrected the cortical-bone defect. CONCLUSIONS: Our study showed that Pyle's disease was caused by a deficiency of sFRP4, that cortical-bone and trabecular-bone homeostasis were governed by different mechanisms, and that sFRP4-mediated cross-regulation between Wnt and BMP signaling was critical for achieving proper cortical-bone thickness and stability. (Funded by the Swiss National Foundation and the National Institutes of Health.).