CAFs-Associated Genes (CAFGs) in Pancreatic Ductal Adenocarcinoma (PDAC) and Novel Therapeutic Strategy.

Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive cancer with striking fibrosis, and its mortality rate is ranked second across human cancers. Cancer-associated fibroblasts (CAFs) play a critical role in PDAC progression, and we reviewed the molecular understanding of PDAC CAFs and novel therapeutic potential at present. CAFs-associated genes (CAFGs) were tentatively classified into three categories by stroma specificity representing stroma/epithelia expression ratios (SE ratios). The recent classification using single cell transcriptome technology clarified that CAFs were composed of myofibroblasts (myCAFs), inflammatory CAFs (iCAFs), and other minor ones (e.g., POSTN-CAFs and antigen presenting CAFs, apCAFs). LRRC15 is a myCAFs marker, and myCAFs depletion by diphtheria toxin induces the rapid accumulation of cytotoxic T lymphocytes (CTLs) and therefore augment PDL1 antibody treatments. This finding proposes that myCAFs may be a critical regulator of tumor immunity in terms of PDAC progression. myCAFs are located in CAFs adjacent to tumor cells, while iCAFs marked by PDPN and/or COL14A1 are distant from tumor cells, where hypoxic and acidic environments being located in iCAFs putatively due to poor blood supply is consistent with HIF1A and GPR68 expressions. iCAFs may be shared with SASP (secretion-associated phenotypes) in senescent CAFs. myCAFs are classically characterized by CAFGs induced by TGFB1, while chemoresistant CAFs with SASP may dependent on IL6 expression and accompanied by STAT3 activation. Recently, it was found that the unique metabolism of CAFs can be targeted to prevent PDAC progression, where PDAC cells utilize glucose, whereas CAFs in turn utilize lactate, which may be epigenetically regulated, mediated by its target genes including CXCR4. In summary, CAFs have unique molecular characteristics, which have been rigorously clarified as novel therapeutic targets of PDAC progression.

Effect of LRRC15 on autophagy in A549 cells.

Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic, and irreversible interstitial lung disease with unknown cause. To explore the role and regulatory mechanism of leucine-rich repeat-containing protein 15 (LRRC15) in IPF, bleomycin (BLM)-induced pulmonary fibrosis in mouse and A549 cells were constructed, and the expression of LRRC15 were detected. Then, MTT, GFP-RFP-LC3 dual fluorescent labeling system and Western blotting were used to investigate the effects of LRRC15 on cell activity and autophagy after transfection of siLRRC15, respectively. The results indicated that the expression of LRRC15 was significantly increased after the BLM treatment in mouse lung tissue and A549 cells. The designed and synthesized siLRRC15 followed by transfection into A549 cells resulted in a dramatic reduction in LRRC15 expression and partially restored the cell damage induced by BLM. Moreover, the expression of LC3-II and P62 were up-regulated, the amount of autophagosome were increased by GFP-RFP-LC3 dual fluorescent labeling assay after BLM treatment. Meanwhile, this study also showed that the key autophagy proteins LC3-II, ATG5 and ATG7 were up-regulated, P62 was down-regulated and autophagic flux were enhanced after further treatment of A549 cells with siLRRC15. The above findings suggest that LRRC15 is an indicator of epithelial cell damage and may participate in the regulation of fibrosis through autophagy mechanism in IPF. This study provides necessary theoretical basis for further elucidating the mechanism of IPF.

Development of Anti-LRRC15 Small Fragments for Imaging Purposes Using a Phage-Display ScFv Approach.

The human leucine-rich repeat-containing protein 15 (LRRC15) is a membrane protein identified as a marker of CAF (cancer-associated fibroblast) cells whose overexpression is positively correlated with cancer grade and outcome. Nuclear molecular imaging (i.e., SPECT and PET) to track LRRC15 expression could be very useful in guiding further therapeutic strategies. In this study, we developed an ScFv mouse phage-display library to obtain small fragment antibodies against human LRRC15 for molecular imaging purposes. Mice were immunized with recombinant human LRRC15 (hLRRC15), and lymph node cells were harvested for ScFv (single-chain variable fragment) phage-display analysis. The built library was used for panning on cell lines with constitutive or induced expression after transfection. The choice of best candidates was performed by screening various other cell lines, using flow cytometry. The selected candidates were reformatted into Cys-ScFv or Cys-diabody by addition of cysteine, and cloned in mammalian expression vectors to obtain batches of small fragments that were further used in site-specific radiolabeling tests. The obtained library was 1.2 × 107 cfu/µg with an insertion rate >95%. The two panning rounds performed on cells permittedenrichment of 2 × 10−3. Screening with flow cytometry allowed us to identify 28 specific hLRRC15 candidates. Among these, two also recognized murine LRCC15 and were reformatted into Cys-ScFv and Cys-diabody. They were expressed transiently in a mammalian system to obtain 1.0 to 4.5 mg of Cys fragments ready for bioconjugation and radiolabeling. Thus, in this paper, we demonstrate the relevance of the phage-display ScFv library approach for the fast-track development of small antibodies for imaging and/or immunotherapy purposes.

Integrated microenvironment-associated genomic profiles identify LRRC15 mediating recurrent glioblastoma-associated macrophages infiltration.

Glioblastoma (GBM) is the most common malignant intracranial tumour with intrinsic infiltrative characteristics, which could lead to most patients eventually relapse. The prognosis of recurrent GBM patients remains unsatisfactory. Cancer cell infiltration and their interaction with the tumour microenvironment (TME) could promote tumour recurrence and treatment resistance. In our study, we aimed to identify potential tumour target correlated with rGBM microenvironment based on the gene expression profiles and clinical information of rGBM patients from The Cancer Genome Atlas (TCGA) database. LRRC15 gene with prognostic value was screened by univariate and multivariate analysis, and the correlation between macrophages and LRRC15 was identified as well. Furthermore, the prognosis correlation and immune characteristics of LRRC15 were validated using the Chinese Glioma Genome Atlas (CGGA) database and our clinical tissues by immunochemistry assay. Additionally, we utilized the transwell assay and carboxy fluorescein succinimidyl ester (CFSE) tracking to further confirm the effects of LRRC15 on attracting microglia/macrophages and tumour cell proliferation in the TME. Gene profiles-based rGBM microenvironment identified that LRRC15 could act in collusion with microglia/macrophages in the rGBM microenvironment to promote the poor prognosis, especially in mesenchymal subtype, indicating the strategies of targeting LRRC15 to improve macrophages-based immunosuppressive effects could be promising for rGBM treatments.

LRRC15 antibody-drug conjugates show promise as osteosarcoma therapeutics in preclinical studies.

BACKGROUND: Osteosarcoma (OS), the most common bone tumor in children and adolescents, has high rates of metastasis leading to poor survival. Leucine-rich repeat containing 15 (LRRC15), a transmembrane protein whose expression is modulated by TGFß, was recently shown to be highly expressed on the surface of OS tumor cells. Here, we evaluate a novel antibody-drug conjugate (ADC) targeting LRRC15 in OS human cell lines and murine xenografts. We compare this new ADC, which is conjugated to the anthracycline derivative PNU-159682 (PNU), to a previously studied LRRC15 ADC that is conjugated to the tubulin inhibitor monomethyl auristatin E (MMAE), since anthracyclines are standard of care in OS. PROCEDURE: We evaluated LRRC15 expression in OS cells using Western blots and flow cytometry, and analyzed the epigenetic landscape of the LRRC15 locus using chromatin immunoprecipitation. Efficacy of ADCs on cell growth was analyzed by IncuCyte live cell imaging. Intramuscular xenograft tumor growth was assessed by bioluminescence imaging and hematoxylin and eosin staining. RESULTS: LRRC15-PNU is more effective at inhibiting growth in vitro and in vivo than an isotype antibody control or the LRRC15-MMAE ADC in two high LRRC15 expressing OS cell lines. Low expressing cell lines are not sensitive to either ADC. Importantly, cells with low LRRC15 expression are amenable to re-expression after TGFß treatment, suggesting a potential to sensitize insensitive OS cells to LRRC15 ADC treatment. In vivo, LRRC15-PNU had cure rates of 40-100% in OS xenograft models. CONCLUSIONS: Overall, LRRC15-directed ADCs are a promising new avenue for OS treatment.

BACH1 changes microglial metabolism and affects astrogenesis during mouse brain development.

Microglia are highly heterogeneous as resident immune cells in the central nervous system. Although the proinflammatory phenotype of microglia is driven by the metabolic transformation in the disease state, the mechanism of metabolic reprogramming in microglia and whether it affects surrounding astrocyte progenitors have not been well elucidated. Here, we illustrate the communication between microglial metabolism and astrogenesis during embryonic development. The transcription factor BTB and CNC homology 1 (Bach1) reduces lactate production by inhibiting two key enzymes, HK2 and GAPDH, during glycolysis. Metabolic perturbation of microglia reduces lactate-dependent histone modification enrichment at the Lrrc15 promoter. The microglia-derived LRRC15 interacts with CD248 to participate in the JAK/STAT pathway and influence astrogenesis. In addition, Bach1cKO-Cx3 mice exhibit abnormal neuronal differentiation and anxiety-like behaviors. Altogether, this work suggests that the maintenance of microglia metabolic homeostasis during early brain development is closely related to astrogenesis, providing insights into astrogenesis and related diseases.

myCAFs are better than yours: targeting myofibroblasts potentiates immunotherapy.

New findings (Krishnamurty et al.) implicate a subset of cancer-associated fibroblasts (CAFs) that express leucine-rich repeat containing 15 (LRRC15) in promoting tumor growth in pancreatic adenocarcinoma (PDAC), by suppressing the antitumor immunity of cytotoxic T cells. Genetic ablation of LRRC15+ CAFs resulted in better response to immune checkpoint blockade, suggesting they may be a novel target for therapy.

RUNX1 Ameliorates Rheumatoid Arthritis Progression through Epigenetic Inhibition of LRRC15.

Leucine-rich repeat containing 15 (LRRC15) has been identified as a contributing factor for cartilage damage in osteoarthritis; however, its involvement in rheumatoid arthritis (RA) and the underlying mechanisms have not been well characterized. The purpose of this study was to explore the function of LRRC15 in RA-associated fibroblast-like synoviocytes (RA-FLS) and in mice with collagen-induced arthritis (CIA) and to dissect the epigenetic mechanisms involved. LRRC15 was overexpressed in the synovial tissues of patients with RA, and LRRC15 overexpression was associated with increased proliferative, migratory, invasive, and angiogenic capacities of RA-FLS and accelerated release of pro-inflammatory cytokines. LRRC15 knockdown significantly inhibited synovial proliferation and reduced bone invasion and destruction in CIA mice. Runt-related transcription factor 1 (RUNX1) transcriptionally represses LRRC15 by binding to core-binding factor subunit beta (CBF-ß). Overexpression of RUNX1 significantly inhibited the invasive phenotype of RA-FLS and suppressed the expression of proinflammatory cytokines. Conversely, the effects of RUNX1 were significantly reversed after overexpression of LRRC15 or inhibition of RUNX1-CBF-ß interactions. Therefore, we demonstrated that RUNX1-mediated transcriptional repression of LRRC15 inhibited the development of RA, which may have therapeutic effects for RA patients.

LRRC superfamily expression in stromal cells predicts the clinical prognosis and platinum resistance of ovarian cancer.

BACKGROUND: Leucine-rich repeat sequence domains are known to mediate protein‒protein interactions. Recently, some studies showed that members of the leucine rich repeat containing (LRRC) protein superfamily may become new targets for the diagnosis and treatment of tumours. However, it is not known whether any of the LRRC superfamily genes is expressed in the stroma of ovarian cancer (OC) and is associated with prognosis. METHODS: The clinical data and transcriptional profiles of OC patients from the public databases TCGA (n = 427), GTEx (n = 88) and GEO (GSE40266 and GSE40595) were analysed by R software. A nomogram model was also generated through R. An online public database was used for auxiliary analysis of prognosis, immune infiltration and protein‒protein interaction (PPI) networks. Immunohistochemistry and qPCR were performed to determine the protein and mRNA levels of genes in high-grade serous ovarian cancer (HGSC) tissues of participants and the MRC-5 cell line induced by TGF-ß. RESULTS: LRRC15 and LRRC32 were identified as differentially expressed genes from the LRRC superfamily by GEO transcriptome analysis. PPI network analysis suggested that they were most enriched in TGF-ß signalling. The TCGA-GTEx analysis results showed that only LRRC15 was highly expressed in both cancer-associated fibroblasts (CAFs) and the tumour stroma of OC and was related to clinical prognosis. Based on this, we developed a nomogram model to predict the incidence of adverse outcomes in OC. Moreover, LRRC15 was positively correlated with CAF infiltration and negatively correlated with CD8 + T-cell infiltration. As a single indicator, LRRC15 had the highest accuracy (AUC = 0.920) in predicting the outcome of primary platinum resistance. CONCLUSIONS: The LRRC superfamily is related to the TGF-ß pathway in the microenvironment of OC. LRRC15, as a stromal biomarker, can predict the clinical prognosis of HGSC and promote the immunosuppressive microenvironment. LRRC15 may be a potential therapeutic target for reversing primary resistance in OC.

The Elusive Coreceptors for the SARS-CoV-2 Spike Protein.

Evidence suggests that the N-terminal domain (NTD) of the SARS-CoV-2 spike protein interacts with host coreceptors that participate in viral entry. Resolving the identity of coreceptors has important clinical implications as it may provide the basis for the development of antiviral drugs and vaccine candidates. The majority of characteristic mutations in variants of concern (VOCs) have occurred in the NTD and receptor binding domain (RBD). Unlike the RBD, mutations in the NTD have clustered in the most flexible parts of the spike protein. Many possible coreceptors have been proposed, including various sugars such as gangliosides, sialosides, and heparan sulfate. Protein coreceptors, including neuropilin-1 and leucine-rich repeat containing 15 (LRRC15), are also proposed coreceptors that engage the NTD.

LRRC15 is an inhibitory receptor blocking SARS-CoV-2 spike-mediated entry in trans.

SARS-CoV-2 infection is mediated by the entry receptor ACE2. Although attachment factors and co-receptors facilitating entry are extensively studied, cellular entry factors inhibiting viral entry are largely unknown. Using a surfaceome CRISPR activation screen, we identified human LRRC15 as an inhibitory receptor for SARS-CoV-2 entry. LRRC15 directly binds to the receptor-binding domain (RBD) of spike protein with a moderate affinity and inhibits spike-mediated entry. Analysis of human lung single cell RNA sequencing dataset reveals that expression of LRRC15 is primarily detected in fibroblasts and particularly enriched in pathological fibroblasts in COVID-19 patients. ACE2 and LRRC15 are not co-expressed in the same cell types in the lung. Strikingly, expression of LRRC15 in ACE2-negative cells blocks spike-mediated viral entry in ACE2+ cell in trans, suggesting a protective role of LRRC15 in a physiological context. Therefore, LRRC15 represents an inhibitory receptor for SARS-CoV-2 regulating viral entry in trans.

LRRC15 Targeting in Soft-Tissue Sarcomas: Biological and Clinical Implications.

BACKGROUND: LRRC15 is a member of the LRR (leucine-rich repeat) superfamily present on tumor-associated fibroblasts (CAFs) and stromal cells. The expression of LRRC15 is upregulated by the pro-inflammatory cytokine TGFß. ABBV-085 is a monomethyl auristatin E (MMAE)-containing antibody-drug conjugate (ADC) designed to target LRRC15, and which has shown significant anti-tumor activity in several tumor models. This is the first focused examination of LRRC15 expression and ABBV-085 activity in soft-tissue sarcomas (STS). METHODS: We analyzed the LRRC15 expression profile by immunohistochemistry in 711 STS cases, covering a broad spectrum of STS histologies and sub-classifications. In vivo experiments were carried out by using LRRC15-positive and LRRC15-negative patient-derived xenograft (PDX) models of STS. RESULTS: In contrast to patterns observed in epithelial tumors, LRRC15 was expressed not only by stromal cells but also by cancer cells in multiple subsets of STS with significant variations noted between histological subtypes. Overexpression of LRRC15 is positively correlated with grade and independently associated with adverse outcome. ABBV-085 has robust preclinical efficacy against LRRC15 positive STS patient-derived xenograft (PDX) models. CONCLUSION: We provide the first preclinical evidence that LRRC15 targeting with an antibody-drug conjugate is a promising strategy in LRRC15-positive STS. ABBV-085 is being evaluated in an ongoing clinical trial in STS and other malignancies.

Expression and clinical implications of leucine-rich repeat containing 15 (LRRC15) in osteosarcoma.

Leucine-rich repeat containing 15 (LRRC15) is a member of the leucine-rich repeat superfamily that is overexpressed in various cancers and associated with higher tumor grade and aggression. Despite its known tumorigenicity, its roles within osteosarcoma are unknown, prompting us to evaluate its expression and clinical significance within this rare yet aggressive cancer. Western blots showed differential expression of LRRC15 in the osteosarcoma cell lines MNNG/HOS, KHOS, 143B, MG63, Saos-2, and U2OS. We additionally validated this positive expression, as well as sublocalization to the cell membrane, with immunofluorescence. A tissue microarray constructed from 69 osteosarcoma patient tissues was immunohistochemically stained for LRRC15 expression, stratified, and used for clinicopathological analysis. Publicly available databases on LRRC15 expression, including RNA sequencing data from the Therapeutically Applicable Research to Generate Effective Treatments on Osteosarcoma (TARGET-OS) and the Gene Expression database of Normal and Tumor tissues 2 (GENT2) were also analyzed. We found 63 of the 69 (91.3%) patient tissues exhibited some degree of LRRC15 immunostaining, including no staining (6 of 69, 8.7%), 1+ staining (12 of 69, 17.4%), 2+ staining (25 of 69, 36.2%), and 3+ staining (26 of 69, 37.7%). The patients with osteosarcomas having elevated LRRC15 expression demonstrated comparatively increased metastasis, chemoresistance, and shorter 5-year survival rates. Our analysis of the TARGET-OS and GENT2 databases also showed increased LRRC15 gene expression in osteosarcoma. Taken together, our study supports LRRC15 as a prognostic biomarker and emerging therapeutic target in osteosarcoma.

LRRC15 promotes osteogenic differentiation of mesenchymal stem cells by modulating p65 cytoplasmic/nuclear translocation.

BACKGROUND: Mesenchymal stem cells (MSCs) are a reliable resource for bone regeneration and tissue engineering, but the molecular mechanisms of differentiation remain unclear. The tumor antigen 15-leucine-rich repeat containing membrane protein (LRRC15) is a transmembrane protein demonstrated to play important roles in cancer. However, little is known about its role in osteogenesis. This study was to evaluate the functions of LRRC15 in osteogenic differentiation of MSCs. METHODS: Osteogenic-induction treatment and the ovariectomized (OVX) model were performed to investigate the potential relationship between LRRC15 and MSC osteogenesis. A loss-of-function study was used to explore the functions of LRRC15 in osteogenic differentiation of MSCs in vitro and in vivo. NF-κB pathway inhibitor BAY117082, siRNA, nucleocytoplasmic separation, and ChIP assays were performed to clarify the molecular mechanism of LRRC15 in bone regulation. RESULTS: Our results first demonstrated that LRRC15 expression was upregulated upon osteogenic induction, and the level of LRRC15 was significantly decreased in OVX mice. Both in-vitro and in-vivo experiments detected that LRRC15 was required for osteogenesis of MSCs. Mechanistically, LRRC15 inhibited transcription factor NF-κB signaling by affecting the subcellular localization of p65. Further studies indicated that LRRC15 regulated osteogenic differentiation in a p65-dependent manner. CONCLUSIONS: Taken together, our findings reveal that LRRC15 is an essential regulator for osteogenesis of MSCs through modulating p65 cytoplasmic/nuclear translocation, and give a novel hint for MSC-mediated bone regeneration.