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 ...

South Australian Health and Medical Research Institute (SAHMRI)

University of Adelaide

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.

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

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  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; <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.

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

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.

Accurate quantification of minimal residual disease (MRD) during treatment of chronic myeloid leukemia (CML) guides clinical decisions. The conventional MRD method, RQ-PCR for  mRNA, reflects a composite of the number of circulating leukemic cells and the  transcripts per cell.  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.  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,  mRNA values declined more rapidly than DNA. By six months, the two measures aligned closely. The expression of  mRNA was normalized to cell number to generate an expression ratio. The expression of e13a2  was lower than that of e14a2 transcripts at multiple time points during treatment.  DNA was quantifiable in 48% of samples with undetectable  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  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  mRNA are proportional to the depletion of leukemic cells.

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

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)(dabpy)] is a potent sensor for NO in its irreversible, NO-bound active form, [Ru(bpy)(T-bpy)]. 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)(dabpy)] 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)(dabpy)] as a sensor to detect changes in NO from human blood samples in cardiovascular health and disease.

Jarrad Goyne

South Australian Health and Medical Research Institute (SAHMRI)

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