Investigation of MIP-1alpha Inhibitory Activity on the CFU-A Stem Cells

Macrophage inflammatory protein-1 alpha (MIP-1alpha) is a member of the chemokine superfamily and has been observed to inhibit the proliferation of transiently engrafting stem cells, namely the colony forming unit-agar (CFU-A) stem cells. This study was initiated to investigate how MIP-1alpha exerts its inhibitory effect on these cells at the genetic level and, to examine whether altering the growth factors required for CFU-A colony formation could interfere with the inhibitory activity of MIP-1alpha. The results presented in this thesis indicate that growth factor alteration has minimal effects on the inhibition of CFU-A colony formation. However, low levels of MIP-1alpha in the presence of high levels of SCF or M-CSF have been observed to stimulate colony formation. This stimulatory activity of MIP-1alpha has been previously observed on progenitor cells however, it has never been reported on stem cells. Furthermore, alternatively shaped CFU-A colonies have been observed in assays containing high levels of both GM-CSF and MIP-1alpha. These results indicate that although the growth factors in the context of this assay can not interfere with the inhibitory signal of MIP-1alpha they may however interact with the other MIP-la signalling pathways. Although in the CFU-A assay SCF and IL-11 could not interfere with the inhibition of CFU-A colonies by MIP-1alpha, it was observed that upon the ex-vivo expansion of bone marrow, with SCF and IL-11, that the inhibitory activity of MIP-1alpha was reduced. This effect was observed to be specific for MIP-1alpha, as TGF-beta inhibition of CFU-A colony formation was not affected, and was proposed to be due to the down regulation of the MIP-1alpha inhibitory receptor. Indeed analysis of MIP-1alpha receptor expression indicated that CCR-1 was up-regulated whereas CCR3 and D6 were both down regulated, however neither CCR-3 nor D6 proved to be involved in MIP-1alpha inhibition of CFU-A colony formation. Therefore this study observed that in the context of the CFU-A assay that MIP-1alpha inhibitory signalling pathway is robust and minimally interacts with SCF, M-CSF, GM-CSF, IL-11 and LIF signalling pathways

Circulating microRNA analysis in a prospective co-clinical trial identifies MIR652-3p as a response biomarker and driver of regorafenib resistance mechanisms in colorectal cancer

Background: The multi-kinase inhibitor regorafenib has demonstrated efficacy in chemo-refractory metastatic colorectal cancer (mCRC) patients. However, lack of predictive biomarkers and concerns over significant toxicities hamper the use of regorafenib in clinical practice. Methods: Serial liquid biopsies were obtained at baseline and monthly until disease progression in chemo-refractory mCRC patients treated with regorafenib in a phase II clinical trial (PROSPECT-R n=40; NCT03010722) and in a multicentric validation cohort (n=241). Tissue biopsies collected at baseline, after 2 months and at progression in the PROSPECT-R trial were used to establish Patient-Derived Organoids (PDOs) and for molecular analyses. MicroRNA profiling was performed on baseline bloods using the NanoString nCounter platform and results were validated by digital droplet PCR and/or In Situ Hybridization in paired liquid and tissue biopsies. PDOs co-cultures and PDO-xenotransplants were generated for functional analyses. Results: Large-scale microRNA expression analysis in longitudinal matched liquid and tissue biopsies from the PROSPECT-R trial identified MIR652-3p as a biomarker of clinical benefit to regorafenib. These findings were confirmed in an independent validation cohort and in a "control" group of 100 patients treated with lonsurf. Using ex vivo co-culture assays paired with single-cell RNA-sequencing of PDO established pre- and post-treatment, we modelled regorafenib response observed in vivo and in patients, and showed that MIR652-3p controls resistance to regorafenib by impairing regorafenib-induced lethal autophagy and by orchestrating the switch from neo-angiogenesis to vessel co-option. Conclusions: Our results identify MIR652-3p as potential biomarker and as a driver of cell and non-cell autonomous mechanisms of resistance to regorafenib