Neuro-mesenchymal interaction mediated by a ß2 adrenergic-nerve growth factor feedforward loop promotes colorectal cancer progression.

Cancer-associated fibroblasts (CAFs) and nerves, components of the tumor microenvironment, have each been shown to directly promote gastrointestinal cancers. However, it remains unknown whether these cells interact with each other to regulate cancer progression. We found that in colorectal cancer (CRC) norepinephrine induces ADRB2-dependent nerve growth factor (NGF) secretion from CAFs, which in turn increases intra-tumor sympathetic innervation and norepinephrine accumulation. Adrenergic stimulation accelerates CRC growth through ADRA2A/Gi-mediated activation of Yes-Associated Protein (YAP). NGF from CAFs directly enhances CRC cell growth via the PI3K/AKT pathway. Treatment with a tropomyosin receptor kinase (Trk) inhibitor decreased YAP and AKT activation and CRC progression in mice. In human CRC, high NGF expression is associated with the mesenchymal-like tumor subtype and poor patient survival. These findings suggest a central role for reciprocal CAF-nerve crosstalk in promoting CRC progression. Blocking this feedforward loop with a Trk inhibitor may represent a potential therapeutic approach for CRC.

Impaired Glycosylation of Gastric Mucins Drives Gastric Tumorigenesis and Serves as a Novel Therapeutic Target.

BACKGROUND & AIMS: Gastric cancer is often accompanied by a loss of mucin 6 (MUC6), but its pathogenic role in gastric carcinogenesis remains unclear. METHODS: Muc6 knockout (Muc6-/-) mice and Muc6-dsRED mice were newly generated. Tff1Cre, Golph3-/-, R26-Golgi-mCherry, Hes1flox/flox, Cosmcflox/flox, and A4gnt-/- mice were also used. Histology, DNA and RNA, proteins, and sugar chains were analyzed by whole-exon DNA sequence, RNA sequence, immunohistochemistry, lectin-binding assays, and liquid chromatography-mass spectrometry analysis. Gastric organoids and cell lines were used for in vitro assays and xenograft experiments. RESULTS: Deletion of Muc6 in mice spontaneously causes pan-gastritis and invasive gastric cancers. Muc6-deficient tumor growth was dependent on mitogen-activated protein kinase activation, mediated by Golgi stress-induced up-regulation of Golgi phosphoprotein 3. Glycomic profiling revealed aberrant expression of mannose-rich N-linked glycans in gastric tumors, detected with banana lectin in association with lack of MUC6 expression. We identified a precursor of clusterin as a binding partner of mannose glycans. Mitogen-activated protein kinase activation, Golgi stress responses, and aberrant mannose expression are found in separate Cosmc- and A4gnt-deficient mouse models that lack normal O-glycosylation. Banana lectin-drug conjugates proved an effective treatment for mannose-rich murine and human gastric cancer. CONCLUSIONS: We propose that Golgi stress responses and aberrant glycans are important drivers of and promising new therapeutic targets for gastric cancer.

Engineering tumor-colonizing E. coli Nissle 1917 for detection and treatment of colorectal neoplasia.

Bioengineered probiotics enable new opportunities to improve colorectal cancer (CRC) screening, prevention and treatment. Here, first, we demonstrate selective colonization of colorectal adenomas after oral delivery of probiotic E. coli Nissle 1917 (EcN) to a genetically-engineered murine model of CRC predisposition and orthotopic models of CRC. We next undertake an interventional, double-blind, dual-centre, prospective clinical trial, in which CRC patients take either placebo or EcN for two weeks prior to resection of neoplastic and adjacent normal colorectal tissue (ACTRN12619000210178). We detect enrichment of EcN in tumor samples over normal tissue from probiotic-treated patients (primary outcome of the trial). Next, we develop early CRC intervention strategies. To detect lesions, we engineer EcN to produce a small molecule, salicylate. Oral delivery of this strain results in increased levels of salicylate in the urine of adenoma-bearing mice, in comparison to healthy controls. To assess therapeutic potential, we engineer EcN to locally release a cytokine, GM-CSF, and blocking nanobodies against PD-L1 and CTLA-4 at the neoplastic site, and demonstrate that oral delivery of this strain reduces adenoma burden by ~50%. Together, these results support the use of EcN as an orally-deliverable platform to detect disease and treat CRC through the production of screening and therapeutic molecules.

Loss of Grem1-lineage chondrogenic progenitor cells causes osteoarthritis.

Osteoarthritis (OA) is characterised by an irreversible degeneration of articular cartilage. Here we show that the BMP-antagonist Gremlin 1 (Grem1) marks a bipotent chondrogenic and osteogenic progenitor cell population within the articular surface. Notably, these progenitors are depleted by injury-induced OA and increasing age. OA is also caused by ablation of Grem1 cells in mice. Transcriptomic and functional analysis in mice found that articular surface Grem1-lineage cells are dependent on Foxo1 and ablation of Foxo1 in Grem1-lineage cells caused OA. FGFR3 signalling was confirmed as a promising therapeutic pathway by administration of pathway activator, FGF18, resulting in Grem1-lineage chondrocyte progenitor cell proliferation, increased cartilage thickness and reduced OA. These findings suggest that OA, in part, is caused by mechanical, developmental or age-related attrition of Grem1 expressing articular cartilage progenitor cells. These cells, and the FGFR3 signalling pathway that sustains them, may be effective future targets for biological management of OA.

Engineered bacteria detect tumor DNA.

Synthetic biology has developed sophisticated cellular biosensors to detect and respond to human disease. However, biosensors have not yet been engineered to detect specific extracellular DNA sequences and mutations. Here, we engineered naturally competent Acinetobacter baylyi to detect donor DNA from the genomes of colorectal cancer (CRC) cells, organoids, and tumors. We characterized the functionality of the biosensors in vitro with coculture assays and then validated them in vivo with sensor bacteria delivered to mice harboring colorectal tumors. We observed horizontal gene transfer from the tumor to the sensor bacteria in our mouse model of CRC. This cellular assay for targeted, CRISPR-discriminated horizontal gene transfer (CATCH) enables the biodetection of specific cell-free DNA.

Advancing translational research for colorectal immuno-oncology.

Colorectal cancer (CRC) is a common and deadly disease. Unfortunately, immune checkpoint inhibitors (ICIs) fail to elicit effective anti-tumour responses in the vast majority of CRC patients. Patients that are most likely to respond are those with DNA mismatch repair deficient (dMMR) and microsatellite instability (MSI) disease. However, reliable predictors of ICI response are lacking, even within the dMMR/MSI subtype. This, together with identification of novel mechanisms to increase response rates and prevent resistance, are ongoing and vitally important unmet needs. To address the current challenges with translation of early research findings into effective therapeutic strategies, this review summarises the present state of preclinical testing used to inform the development of immuno-regulatory treatment strategies for CRC. The shortfalls and advantages of commonly utilised mouse models of CRC, including chemically induced, transplant and transgenic approaches are highlighted. Appropriate use of existing models, incorporation of patient-derived data and development of cutting-edge models that recapitulate important features of human disease will be key to accelerating clinically relevant research in this area.

Loss of Grem1-articular cartilage progenitor cells causes osteoarthritis.

Osteoarthritis (OA), which carries an enormous disease burden across the world, is characterised by irreversible degeneration of articular cartilage (AC), and subsequently bone. The cellular cause of OA is unknown. Here, using lineage tracing in mice, we show that the BMP-antagonist Gremlin 1 (Grem1) marks a novel chondrogenic progenitor (CP) cell population in the articular surface that generates joint cartilage and subchondral bone during development and adulthood. Notably, this CP population is depleted in injury-induced OA, and with age. OA is also induced by toxin-mediated ablation of Grem1 CP cells in young mice. Transcriptomic analysis and functional modelling in mice revealed articular surface Grem1-lineage cells are dependent on Foxo1; ablation of Foxo1 in Grem1-lineage cells led to early OA. This analysis identified FGFR3 signalling as a therapeutic target, and injection of its activator, FGF18, caused proliferation of Grem1-lineage CP cells, increased cartilage thickness, and reduced OA pathology. We propose that OA arises from the loss of CP cells at the articular surface secondary to an imbalance in progenitor cell homeostasis and present a new progenitor population as a locus for OA therapy.

Colorectal cancer detection and treatment with engineered probiotics.

Bioengineered probiotics enable new opportunities to improve colorectal cancer (CRC) screening, prevention and treatment strategies. Here, we demonstrate the phenomenon of selective, long-term colonization of colorectal adenomas after oral delivery of probiotic E. coli Nissle 1917 (EcN) to a genetically-engineered murine model of CRC predisposition. We show that, after oral administration, adenomas can be monitored over time by recovering EcN from stool. We also demonstrate specific colonization of EcN to solitary neoplastic lesions in an orthotopic murine model of CRC. We then exploit this neoplasia-homing property of EcN to develop early CRC intervention strategies. To detect lesions, we engineer EcN to produce a small molecule, salicylate, and demonstrate that oral delivery of this strain results in significantly increased levels of salicylate in the urine of adenoma-bearing mice, in comparison to healthy controls. We also assess EcN engineered to locally release immunotherapeutics at the neoplastic site. Oral delivery to mice bearing adenomas, reduced adenoma burden by ∻50%, with notable differences in the spatial distribution of T cell populations within diseased and healthy intestinal tissue, suggesting local induction of robust anti-tumor immunity. Together, these results support the use of EcN as an orally-delivered platform to detect disease and treat CRC through its production of screening and therapeutic molecules.

Non-Helicobacter pylori Gastric Microbiome Modulates Prooncogenic Responses and Is Associated With Gastric Cancer Risk.

Background and Aims: Although Helicobacter pylori is the most important bacterial carcinogen in gastric cancer (GC), GC can emerge even after H. pylori eradication. Studies suggest that various constituents of the gastric microbiome may influence GC development, but the role of individual pathogens is unclear. Methods: Human gastric mucosal samples were analyzed by 16SrRNA sequencing to investigate microbiome composition and its association with clinical parameters, including GC risk. Identified bacteria in the stomach were cocultured with gastric epithelial cells or inoculated into mice, and transcriptomic changes, DNA damage, and inflammation were analyzed. Bacterial reads in GC tissues were examined together with transcriptomic and genetic sequencing data in the cancer genome atlas dataset. Results: Patients after Helicobacter pylori eradication formed 3 subgroups based on the microbial composition revealed by 16SrRNA sequencing. One dysbiotic group enriched with Fusobacterium and Neisseria species was associated with a significantly higher GC incidence. These species activated prooncogenic pathways in gastric epithelial cells and promoted inflammation in mouse stomachs. Sugar chains that constitute gastric mucin attenuate host-bacteria interactions. Metabolites from Fusobacterium species were genotoxic, and the presence of the bacteria was associated with an inflammatory signature and a higher tumor mutation burden. Conclusion: Gastric microbiota in the dysbiotic stomach is associated with GC development after H. pylori eradication and plays a pathogenic role through direct host-bacteria interaction.

The Antianginal Drug Perhexiline Displays Cytotoxicity against Colorectal Cancer Cells In Vitro: A Potential for Drug Repurposing.

Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide. Perhexiline, a prophylactic anti-anginal drug, has been reported to have anti-tumour effects both in vitro and in vivo. Perhexiline as used clinically is a 50:50 racemic mixture ((R)-P) of (-) and (+) enantiomers. It is not known if the enantiomers differ in terms of their effects on cancer. In this study, we examined the cytotoxic capacity of perhexiline and its enantiomers ((-)-P and (+)-P) on CRC cell lines, grown as monolayers or spheroids, and patient-derived organoids. Treatment of CRC cell lines with (R)-P, (-)-P or (+)-P reduced cell viability, with IC50 values of ~4 µM. Treatment was associated with an increase in annexin V staining and caspase 3/7 activation, indicating apoptosis induction. Caspase 3/7 activation and loss of structural integrity were also observed in CRC cell lines grown as spheroids. Drug treatment at clinically relevant concentrations significantly reduced the viability of patient-derived CRC organoids. Given these in vitro findings, perhexiline, as a racemic mixture or its enantiomers, warrants further investigation as a repurposed drug for use in the management of CRC.

The Origin and Contribution of Cancer-Associated Fibroblasts in Colorectal Carcinogenesis.

BACKGROUND & AIMS: Cancer-associated fibroblasts (CAFs) play an important role in colorectal cancer (CRC) progression and predict poor prognosis in CRC patients. However, the cellular origins of CAFs remain unknown, making it challenging to therapeutically target these cells. Here, we aimed to identify the origins and contribution of colorectal CAFs associated with poor prognosis. METHODS: To elucidate CAF origins, we used a colitis-associated CRC mouse model in 5 different fate-mapping mouse lines with 5-bromodeoxyuridine dosing. RNA sequencing of fluorescence-activated cell sorting-purified CRC CAFs was performed to identify a potential therapeutic target in CAFs. To examine the prognostic significance of the stromal target, CRC patient RNA sequencing data and tissue microarray were used. CRC organoids were injected into the colons of knockout mice to assess the mechanism by which the stromal gene contributes to colorectal tumorigenesis. RESULTS: Our lineage-tracing studies revealed that in CRC, many ACTA2+ CAFs emerge through proliferation from intestinal pericryptal leptin receptor (Lepr)+ cells. These Lepr-lineage CAFs, in turn, express melanoma cell adhesion molecule (MCAM), a CRC stroma-specific marker that we identified with the use of RNA sequencing. High MCAM expression induced by transforming growth factor ß was inversely associated with patient survival in human CRC. In mice, stromal Mcam knockout attenuated orthotopically injected colorectal tumoroid growth and improved survival through decreased tumor-associated macrophage recruitment. Mechanistically, fibroblast MCAM interacted with interleukin-1 receptor 1 to augment nuclear factor κB-IL34/CCL8 signaling that promotes macrophage chemotaxis. CONCLUSIONS: In colorectal carcinogenesis, pericryptal Lepr-lineage cells proliferate to generate MCAM+ CAFs that shape the tumor-promoting immune microenvironment. Preventing the expansion/differentiation of Lepr-lineage CAFs or inhibiting MCAM activity could be effective therapeutic approaches for CRC.

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma.

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment, play a crucial role in metastatic CRC progression and predict poor patient prognosis. However, there is a lack of satisfactory mouse models to study the crosstalk between metastatic cancer cells and CAFs. Here, we present a method to investigate how liver metastasis progression is regulated by the metastatic niche and possibly could be restrained by stroma-directed therapy. Portal vein injection of CRC organoids generated a desmoplastic reaction, which faithfully recapitulated the fibroblast-rich histology of human CRC liver metastases. This model was tissue-specific with a higher tumor burden in the liver when compared to an intra-splenic injection model, simplifying mouse survival analyses. By injecting luciferase-expressing tumor organoids, tumor growth kinetics could be monitored by in vivo imaging. Moreover, this preclinical model provides a useful platform to assess the efficacy of therapeutics targeting the tumor mesenchyme. We describe methods to examine whether adeno-associated virus-mediated delivery of a tumor-inhibiting stromal gene to hepatocytes could remodel the tumor microenvironment and improve mouse survival. This approach enables the development and assessment of novel therapeutic strategies to inhibit hepatic metastasis of CRC.

The BMP antagonist gremlin 1 contributes to the development of cortical excitatory neurons, motor balance and fear responses.

Bone morphogenetic protein (BMP) signaling is required for early forebrain development and cortical formation. How the endogenous modulators of BMP signaling regulate the structural and functional maturation of the developing brain remains unclear. Here, we show that expression of the BMP antagonist Grem1 marks committed layer V and VI glutamatergic neurons in the embryonic mouse brain. Lineage tracing of Grem1-expressing cells in the embryonic brain was examined by administration of tamoxifen to pregnant Grem1creERT; Rosa26LSLTdtomato mice at 13.5 days post coitum (dpc), followed by collection of embryos later in gestation. In addition, at 14.5 dpc, bulk mRNA-seq analysis of differentially expressed transcripts between FACS-sorted Grem1-positive and -negative cells was performed. We also generated Emx1-cre-mediated Grem1 conditional knockout mice (Emx1-Cre;Grem1flox/flox) in which the Grem1 gene was deleted specifically in the dorsal telencephalon. Grem1Emx1cKO animals had reduced cortical thickness, especially layers V and VI, and impaired motor balance and fear sensitivity compared with littermate controls. This study has revealed new roles for Grem1 in the structural and functional maturation of the developing cortex.

Delineating proinflammatory microenvironmental signals by ex vivo modeling of the immature intestinal stroma.

The intestinal stroma provides an important microenvironment for immune cell activation. The perturbation of this tightly regulated process can lead to excessive inflammation. We know that upregulated Toll-like receptor 4 (TLR4) in the intestinal epithelium plays a key role in the inflammatory condition of preterm infants, such as necrotizing enterocolitis (NEC). However, the surrounding stromal contribution to excessive inflammation in the pre-term setting awaits careful dissection. Ex vivo co-culture of embryonic day 14.5 (E14.5) or adult murine intestinal stromal cells with exogenous monocytes was undertaken. We also performed mRNAseq analysis of embryonic and adult stromal cells treated with vehicle control or lipopolysaccharide (LPS), followed by pathway and network analyses of differentially regulated transcripts. Cell characteristics were compared using flow cytometry and pHrodo red phagocytic stain, candidate gene analysis was performed via siRNA knockdown and gene expression measured by qPCR and ELISA. Embryonic stromal cells promote the differentiation of co-cultured monocytes to CD11bhighCD11chigh mononuclear phagocytes, that in turn express decreased levels of CD103. Global mRNAseq analysis of stromal cells following LPS stimulation identified TLR signaling components as the most differentially expressed transcripts in the immature compared to adult setting. We show that CD14 expressed by CD11b+CD45+ embryonic stromal cells is a key inducer of TLR mediated inflammatory cytokine production and phagocytic activity of monocyte derived cells. We utilise transcriptomic analyses and functional ex vivo modelling to improve our understanding of unique molecular cues provided by the immature intestinal stroma.

Stromal DLK1 promotes proliferation and inhibits differentiation of the intestinal epithelium during development.

The stem/progenitor cells of the developing intestine are biologically distinct from their adult counterparts. Here, we examine the microenvironmental cues that regulate the embryonic stem/progenitor population, focusing on the role of Notch pathway factor delta-like protein-1 (DLK1). mRNA-seq analyses of intestinal mesenchymal cells (IMCs) collected from embryonic day 14.5 (E14.5) or adult IMCs and a novel coculture system with E14.5 intestinal epithelial organoids were used. Following addition of recombinant DLK1 (rDLK) or Dlk1 siRNA (siDlk1), epithelial characteristics were compared using imaging, replating efficiency assays, qPCR, and immunocytochemistry. The intestinal phenotypes of littermate Dlk1+/+ and Dlk1-/- mice were compared using immunohistochemistry. Using transcriptomic analyses, we identified morphogens derived from the embryonic mesenchyme that potentially regulate the developing epithelial cells, to focus on Notch family candidate DLK1. Immunohistochemistry indicated that DLK1 was expressed exclusively in the intestinal stroma at E14.5 at the top of emerging villi, decreased after birth, and shifted to the intestinal epithelium in adulthood. In coculture experiments, addition of rDLK1 to adult IMCs inhibited organoid differentiation, whereas Dlk1 knockdown in embryonic IMCs increased epithelial differentiation to secretory lineage cells. Dlk1-/- mice had restricted Ki67+ cells in the villi base and increased secretory lineage cells compared with Dlk1+/+ embryos. Mesenchyme-derived DLK1 plays an important role in the promotion of epithelial stem/precursor expansion and prevention of differentiation to secretory lineages in the developing intestine.NEW & NOTEWORTHY Using a novel coculture system, transcriptomics, and transgenic mice, we investigated differential molecular signaling between the intestinal epithelium and mesenchyme during development and in the adult. We show that the Notch pathway factor delta-like protein-1 (DLK1) is stromally produced during development and uncover a new role for DLK1 in the regulation of intestinal epithelial stem/precursor expansion and differentiation to secretory lineages.

Young-onset colorectal cancer is associated with a personal history of type 2 diabetes.

BACKGROUND: Colorectal cancer (CRC) is rising in incidence in young adults, and this observation is currently unexplained. We investigated whether having a personal history of type 2 diabetes mellitus (T2D) was a potential risk factor for young-onset colorectal cancer (YOCRC). METHODS: The South Australian Young Onset (SAYO) CRC study is a series of young adults with CRC below age 55. Ninety unrelated YOCRC cases were recruited to the study. Personal history and detailed family history of T2D were obtained at face-to-face interview and confirmed from medical records. Whole exome sequencing was conducted on germline DNA from each CRC case. Controls for personal history studies of T2D were 240 patients with proven clear colonoscopies and no known CRC predispositions. RESULTS: The median age of YOCRC cases was 44 years (18-54) and of controls was 45 years (18-54), and 53% of both cases and controls were females (P = 0.99). Left-sided (distal) CRC was seen in 67/89 (75%) of cases. A personal history of T2D was confirmed in 17/90 (19%) YOCRC patients compared with controls (12/240, 5%; P < 0.001; odds ratio = 4.4; 95% confidence interval, 2.0-9.7). YOCRC patients frequently reported at least one first-degree relative with T2D (32/85, 38%). Ten of 87 (12%) of YOCRC cases had CRC-related pathogenic germline variants, however, no pathogenic variants in familial diabetes-associated genes were seen. CONCLUSIONS: Though the mechanism remains unclear, our observations suggest that there is enrichment for personal history of T2D in YOCRC patients. IMPACT: A diagnosis of T2D could therefore potentially identify a subset of young adults at increased risk for CRC and in whom early screening might be appropriate.

The Balance of Stromal BMP Signaling Mediated by GREM1 and ISLR Drives Colorectal Carcinogenesis.

BACKGROUND & AIMS: Cancer-associated fibroblasts (CAFs), key constituents of the tumor microenvironment, either promote or restrain tumor growth. Attempts to therapeutically target CAFs have been hampered by our incomplete understanding of these functionally heterogeneous cells. Key growth factors in the intestinal epithelial niche, bone morphogenetic proteins (BMPs), also play a critical role in colorectal cancer (CRC) progression. However, the crucial proteins regulating stromal BMP balance and the potential application of BMP signaling to manage CRC remain largely unexplored. METHODS: Using human CRC RNA expression data, we identified CAF-specific factors involved in BMP signaling, then verified and characterized their expression in the CRC stroma by in situ hybridization. CRC tumoroids and a mouse model of CRC hepatic metastasis were used to test approaches to modify BMP signaling and treat CRC. RESULTS: We identified Grem1 and Islr as CAF-specific genes involved in BMP signaling. Functionally, GREM1 and ISLR acted to inhibit and promote BMP signaling, respectively. Grem1 and Islr marked distinct fibroblast subpopulations and were differentially regulated by transforming growth factor ß and FOXL1, providing an underlying mechanism to explain fibroblast biological dichotomy. In patients with CRC, high GREM1 and ISLR expression levels were associated with poor and favorable survival, respectively. A GREM1-neutralizing antibody or fibroblast Islr overexpression reduced CRC tumoroid growth and promoted Lgr5+ intestinal stem cell differentiation. Finally, adeno-associated virus 8 (AAV8)-mediated delivery of Islr to hepatocytes increased BMP signaling and improved survival in our mouse model of hepatic metastasis. CONCLUSIONS: Stromal BMP signaling predicts and modifies CRC progression and survival, and it can be therapeutically targeted by novel AAV-directed gene delivery to the liver.

Targeted Disruption of Bone Marrow Stromal Cell-Derived Gremlin1 Limits Multiple Myeloma Disease Progression In Vivo.

In most instances, multiple myeloma (MM) plasma cells (PCs) are reliant on factors made by cells of the bone marrow (BM) stroma for their survival and growth. To date, the nature and cellular composition of the BM tumor microenvironment and the critical factors which drive tumor progression remain imprecisely defined. Our studies show that Gremlin1 (Grem1), a highly conserved protein, which is abundantly secreted by a subset of BM mesenchymal stromal cells, plays a critical role in MM disease development. Analysis of human and mouse BM stromal samples by quantitative PCR showed that GREM1/Grem1 expression was significantly higher in the MM tumor-bearing cohorts compared to healthy controls (p < 0.05, Mann-Whitney test). Additionally, BM-stromal cells cultured with 5TGM1 MM PC line expressed significantly higher levels of Grem1, compared to stromal cells alone (p < 0.01, t-test), suggesting that MM PCs promote increased Grem1 expression in stromal cells. Furthermore, the proliferation of 5TGM1 MM PCs was found to be significantly increased when co-cultured with Grem1-overexpressing stromal cells (p < 0.01, t-test). To examine the role of Grem1 in MM disease in vivo, we utilized the 5TGM1/KaLwRij mouse model of MM. Our studies showed that, compared to immunoglobulin G (IgG) control antibody-treated mice, mice treated with an anti-Grem1 neutralizing antibody had a decrease in MM tumor burden of up to 81.2% (p < 0.05, two-way ANOVA). The studies presented here demonstrate, for the first time, a novel positive feedback loop between MM PCs and BM stroma, and that inhibiting this vicious cycle with a neutralizing antibody can dramatically reduce tumor burden in a preclinical mouse model of MM.