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.

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.

Biological Sensing of Nitric Oxide in Macrophages and Atherosclerosis Using a Ruthenium-Based Sensor.

Macrophage-derived nitric oxide (NO) plays a critical role in atherosclerosis and presents as a potential biomarker. We assessed the uptake, distribution, and NO detection capacity of an irreversible, ruthenium-based, fluorescent NO sensor (Ru-NO) in macrophages, plasma, and atherosclerotic plaques. In vitro, incubation of Ru-NO with human THP1 monocytes and THP1-PMA macrophages caused robust uptake, detected by Ru-NO fluorescence using mass-cytometry, confocal microscopy, and flow cytometry. THP1-PMA macrophages had higher Ru-NO uptake (+13%, p < 0.05) than THP1 monocytes with increased Ru-NO fluorescence following lipopolysaccharide stimulation (+14%, p < 0.05). In mice, intraperitoneal infusion of Ru-NO found Ru-NO uptake was greater in peritoneal CD11b+F4/80+ macrophages (+61%, p < 0.01) than CD11b+F4/80− monocytes. Infusion of Ru-NO into Apoe−/− mice fed high-cholesterol diet (HCD) revealed Ru-NO fluorescence co-localised with atherosclerotic plaque macrophages. When Ru-NO was added ex vivo to aortic cell suspensions from Apoe−/− mice, macrophage-specific uptake of Ru-NO was demonstrated. Ru-NO was added ex vivo to tail-vein blood samples collected monthly from Apoe−/− mice on HCD or chow. The plasma Ru-NO fluorescence signal was higher in HCD than chow-fed mice after 12 weeks (37.9%, p < 0.05). Finally, Ru-NO was added to plasma from patients (N = 50) following clinically-indicated angiograms. There was lower Ru-NO fluorescence from plasma from patients with myocardial infarction (−30.7%, p < 0.01) than those with stable coronary atherosclerosis. In conclusion, Ru-NO is internalised by macrophages in vitro, ex vivo, and in vivo, can be detected in atherosclerotic plaques, and generates measurable changes in fluorescence in murine and human plasma. Ru-NO displays promising utility as a sensor of atherosclerosis.

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.

A Novel Ruthenium-based Molecular Sensor to Detect Endothelial Nitric Oxide.

Nitric oxide (NO) is a key regulator of endothelial cell and vascular function. The direct measurement of NO is challenging due to its short half-life, and as such surrogate measurements are typically used to approximate its relative concentrations. Here we demonstrate that ruthenium-based [Ru(bpy)2(dabpy)]2+ is a potent sensor for NO in its irreversible, NO-bound active form, [Ru(bpy)2(T-bpy)]2+. Using spectrophotometry we established the sensor's ability to detect and measure soluble NO in a concentration-dependent manner in cell-free media. Endothelial cells cultured with acetylcholine or hydrogen peroxide to induce endogenous NO production showed modest increases of 7.3 ± 7.1% and 36.3 ± 25.0% respectively in fluorescence signal from baseline state, while addition of exogenous NO increased their fluorescence by 5.2-fold. The changes in fluorescence signal were proportionate and comparable against conventional NO assays. Rabbit blood samples immediately exposed to [Ru(bpy)2(dabpy)]2+ displayed 8-fold higher mean fluorescence, relative to blood without sensor. Approximately 14% of the observed signal was NO/NO adduct-specific. Optimal readings were obtained when sensor was added to freshly collected blood, remaining stable during subsequent freeze-thaw cycles. Clinical studies are now required to test the utility of [Ru(bpy)2(dabpy)]2+ as a sensor to detect changes in NO from human blood samples in cardiovascular health and disease.

BCR-ABL1 genomic DNA PCR response kinetics during first-line imatinib treatment of chronic myeloid leukemia.

Accurate quantification of minimal residual disease (MRD) during treatment of chronic myeloid leukemia (CML) guides clinical decisions. The conventional MRD method, RQ-PCR for BCR-ABL1 mRNA, reflects a composite of the number of circulating leukemic cells and the BCR-ABL1 transcripts per cell. BCR-ABL1 genomic DNA only reflects leukemic cell number. We used both methods in parallel to determine the relative contribution of the leukemic cell number to molecular response. BCR-ABL1 DNA PCR and RQ-PCR were monitored up to 24 months in 516 paired samples from 59 newly-diagnosed patients treated with first-line imatinib in the TIDEL-II study. In the first three months of treatment, BCR-ABL1 mRNA values declined more rapidly than DNA. By six months, the two measures aligned closely. The expression of BCR-ABL1 mRNA was normalized to cell number to generate an expression ratio. The expression of e13a2 BCR-ABL1 was lower than that of e14a2 transcripts at multiple time points during treatment. BCR-ABL1 DNA was quantifiable in 48% of samples with undetectable BCR-ABL1 mRNA, resulting in MRD being quantifiable for an additional 5-18 months (median 12 months). These parallel studies show for the first time that the rapid decline in BCR-ABL1 mRNA over the first three months of treatment is due to a reduction in both cell number and transcript level per cell, whereas beyond three months, falling levels of BCR-ABL1 mRNA are proportional to the depletion of leukemic cells.

ABCC6 plays a significant role in the transport of nilotinib and dasatinib, and contributes to TKI resistance in vitro, in both cell lines and primary patient mononuclear cells.

ATP Binding Cassette family efflux proteins ABCB1 and ABCG2 have previously been demonstrated to interact with Tyrosine Kinase Inhibitors (TKIs); however, evidence for the interaction of other potentially relevant drug transporters with TKIs is lacking. Through Taqman transporter array technology we assessed the impact of nilotinib on mRNA expression of ABC transporters, with ABCC6 identified as a transporter of interest. Additionally, increased expression of ABCC6 mRNA was observed during in vitro development of nilotinib resistance in BCR-ABL1-expressing cell lines. K562 cells exposed to gradually increasing concentrations of nilotinib (to 2 µM) expressed up to 57-fold higher levels of ABCC6 mRNA when compared with control cells (p = 0.002). Analogous results were observed in nilotinib resistant K562-Dox cells (up to 33-fold higher levels of ABCC6, p = 0.002). IC50 experiments were conducted on patient mononuclear cells in the absence and presence of three ABCC6 inhibitors: indomethacin, probenecid and pantoprazole. Results demonstrated that all three inhibitors significantly reduced nilotinib IC50 (p<0.001) indicating ABCC6 is likely involved in nilotinib transport. Cell line data confirmed these findings. Similar results were obtained for dasatinib, but not imatinib. Combined, these studies suggest that nilotinib and dasatinib are likely substrates of ABCC6 and to our knowledge, this is the first report of ABCC6 involvement in TKI transport. In addition, ABCC6 overexpression may also contribute to nilotinib and dasatinib resistance in vitro. With nilotinib and dasatinib now front line therapy options in the treatment of CML, concomitant administration of ABCC6 inhibitors may present an attractive option to enhance TKI efficacy.

Increased peroxisome proliferator-activated receptor γ activity reduces imatinib uptake and efficacy in chronic myeloid leukemia mononuclear cells.

Imatinib is actively transported by organic cation transporter-1 (OCT-1) influx transporter, and low OCT-1 activity in diagnostic chronic myeloid leukemia blood mononuclear cells is significantly associated with poor molecular response to imatinib. Herein we report that, in diagnostic chronic myeloid leukemia mononuclear cells and BCR-ABL1+ cell lines, peroxisome proliferator-activated receptor γ agonists (GW1929, rosiglitazone, pioglitazone) significantly decrease OCT-1 activity; conversely, peroxisome proliferator-activated receptor γ antagonists (GW9662, T0070907) increase OCT-1 activity. Importantly, these effects can lead to corresponding changes in sensitivity to BCR-ABL kinase inhibition. Results were confirmed in peroxisome proliferator-activated receptor γ-transduced K562 cells. Furthermore, we identified a strong negative correlation between OCT-1 activity and peroxisome proliferator-activated receptor γ transcriptional activity in diagnostic chronic myeloid leukemia patients (n=84; P<0.0001), suggesting that peroxisome proliferator-activated receptor γ activation has a negative impact on the intracellular uptake of imatinib and consequent BCR-ABL kinase inhibition. The inter-patient variability of peroxisome proliferator-activated receptor γ activation likely accounts for the heterogeneity observed in patient OCT-1 activity at diagnosis. Recently, the peroxisome proliferator-activated receptor γ agonist pioglitazone was reported to act synergistically with imatinib, targeting the residual chronic myeloid leukemia stem cell pool. Our findings suggest that peroxisome proliferator-activated receptor γ ligands have differential effects on circulating mononuclear cells compared to stem cells. Since the effect of peroxisome proliferator-activated receptor γ activation on imatinib uptake in mononuclear cells may counteract the clinical benefit of this activation in stem cells, caution should be applied when combining these therapies, especially in patients with high peroxisome proliferator-activated receptor γ transcriptional activity.

Safety and efficacy of imatinib cessation for CML patients with stable undetectable minimal residual disease: results from the TWISTER study.

Most patients with chronic myeloid leukemia (CML) treated with imatinib will relapse if treatment is withdrawn. We conducted a prospective clinical trial of imatinib withdrawal in 40 chronic-phase CML patients who had sustained undetectable minimal residual disease (UMRD) by conventional quantitative polymerase chain reaction (PCR) on imatinib for at least 2 years. Patients stopped imatinib and were monitored frequently for molecular relapse. At 24 months, the actuarial estimate of stable treatment-free remission was 47.1%. Most relapses occurred within 4 months of stopping imatinib, and no relapses beyond 27 months were seen. In the 21 patients treated with interferon before imatinib, a shorter duration of interferon treatment before imatinib was significantly associated with relapse risk, as was slower achievement of UMRD after switching to imatinib. Highly sensitive patient-specific BCR-ABL DNA PCR showed persistence of the original CML clone in all patients with stable UMRD, even several years after imatinib withdrawal. No patients with molecular relapse after discontinuation have progressed or developed BCR-ABL mutations (median follow-up, 42 months). All patients who relapsed remained sensitive to imatinib re-treatment. These results confirm the safety and efficacy of a trial of imatinib withdrawal in stable UMRD with frequent, sensitive molecular monitoring and early rescue of molecular relapse.

Durable complete molecular remission of chronic myeloid leukemia following dasatinib cessation, despite adverse disease features.

Patients with chronic myeloid leukemia, treated with imatinib, who have a durable complete molecular response, might remain in complete molecular response after stopping treatment. Previous reports of patients stopping treatment in complete molecular response have included only patients with a good response to imatinib. We describe 3 patients with stable complete molecular response on dasatinib treatment following imatinib failure. Two of the 3 patients remain in complete molecular response more than 12 months after stopping dasatinib. In these 2 patients we used highly sensitive patient-specific BCR-ABL1 DNA PCR to show that the leukemic clone remains detectable, as we have previously shown in imatinib-treated patients. Dasatinib-associated immunological phenomena, such as the emergence of clonal T-cell populations, were observed both in one patient who relapsed and in one patient in remission. Our results suggest that the characteristics of complete molecular response on dasatinib treatment may be similar to that achieved with imatinib, at least in patients with adverse disease features.