Targeting cancer stem cell plasticity to overcome colorectal cancer resistance and relapse
Introduction
Tumor relapse in colorectal cancer (CRC) may be attributed to an immortal cancer cell population known as cancer stem cells (CSC). These tumor cells potentiate metastatic progression by exploiting their high capacity to self-renew and differentiate. Moreover, CSCs exhibit plasticity, allowing cells to alter their phenotype in response to environmental cues that bolster inherent drug resistance. Given these unique properties, CSCs are an attractive drug target. Our group and others have previously attempted targeting CSCs using antibody-drug conjugates (ADCs) against the Leucine-rich repeat-containing G protein-coupled Receptor 5 (LGR5), a frequently upregulated colorectal CSC marker. Our attempts at eradicating CRC by therapeutically targeting LGR5-positive (LGR5+) CSCs with an LGR5-directed ADC promoted an initial regression. However, some tumors relapsed following treatment completion. Follow-up studies suggest that colorectal cancer cells evade this therapeutic insult by converting into a quiescent LGR5 negative (LGR5-) state. Further, the LGR5- drug-resistant cells use the MET-STAT3 signaling cascade to bolster their invasive and metastatic potential. The long-term goals of this study include elucidating the intracellular players fueling LGR5+ CSC plasticity and developing a dual-targeting therapeutic strategy to overcome CSC-driven tumor heterogeneity.
Methods
We explored the mechanistic relationship between MET-STAT3 and LGR5 through MET or LGR5 ablation and investigated the effects of LGR5 deletion on downstream signaling via co-immunoprecipitation (co-IP) assays. Further, we have cloned two anti-MET monoclonal antibodies (mAbs). Once produced, the specificity and binding affinity of the anti-MET mAbs was evaluated in vitro. The lead mAb candidate was conjugated to the cytotoxic drug payloads. Efficacy of MET- and LGR5-ADCs alone or in combination will be determined ex vivo and in vivo.
Results
Through a co-IP assay, we found increased interaction between MET and IQGAP1 in LGR5 knockdown (KD) cells. Using patented sequences, we successfully cloned, produced, and purified therapeutic MET mAbs. Immunocytochemistry (ICC) and confocal microscopy revealed that both MET mAbs exhibit a high affinity and internalization in CRC cells. MET and LGR5 ADCs were generated by attaching the lead MET mAb to the cytotoxic payload pyrrolobenzodiazepine (PBD) via site-directed conjugation. Proof of concept cytotoxicity assays revealed that our MET ADC exhibited significant potency in vitro.
Conclusion
This study will help understand the regulatory roles of MET and LGR5 in CRC plasticity. Furthermore, the high tumor expression of LGR5 and MET in CRC makes them ideal ADC targets. To overcome tumor heterogeneity and plasticity, ADC-mediated co-targeting of LGR5+ CSCs and their LGR5- counterparts hold strong promise to improve CRC patient prognosis and survival significantly.