Periodic Reporting for period 2 - residualCRC (MECHANISMS BEHIND RESIDUAL DISEASE IN COLORECTAL CANCER and MODELLING OF THERAPIES THAT PREVENT RELAPSE) Reporting period: 2022-03-01 to 2023-08-31 Summary of the context and overall objectives of the project Surgical removal of the primary tumor is part of the standard of care for patients with stage I-III colorectal cancer (CRC). However, despite this treatment, 30-40% of these patients undergo metastatic relapse in the following years, which is a significant cause of death. Unfortunately, the study of metastatic recurrence in CRC has been largely neglected due to the challenges associated with investigating clinically undetectable residual disease and a lack of appropriate preclinical models. In this ERC grant proposal, we aim to address this gap by utilizing novel CRC relapse models developed in my laboratory. We have identified a specific cell type, which we have named HRCs (High Relapse Cells) that seeds the metastatic site in humans and mouse primary tumors. HRCs are responsible for forming residual micrometastatic lesions that later regenerate the disease in foreign organs. Notably, these micrometastases lack an immune protective tumor microenvironment and are infiltrated by immune cells. Accordingly, we provide proof of concept that neoadjuvant immunotherapy can eradicate HRCs and prevent disease relapse. In addition, we are making substantial advances to understand how residual tumor cells resist chemotherapy and regenerate the disease after therapy. Our ultimate goal is to design new therapies that prevent disease relapse. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The human-like colorectal cancer relapse models developed in my laboratory have enabled the study of metastatic dynamics during disease relapse. By means of sequencing at the single cell level micrometastases, small metastases and large metastases, we have identified a cell population occult in mouse livers after primary CRC surgery, which we have termed HRCs (High Relapse Cells). HRCs are also abundant at invasion fronts in human and mouse primary tumors, they retain an epithelial program and express genes involved in cell adhesion, locomotion and extracellular matrix remodeling. Genetic ablation of HRCs prior to extirpation of the primary CRC prevented metastatic recurrence and mice remained disease-free after surgery. Importantly, we demonstrated that micrometastases are immune infiltrated and that neoadyuvant immunotherapy with checkpoint blockers (anti PD-1; anti CTL4) can prevent disease relapse in our experimental models. Our findings, published in the journal Nature (Cañellas-Socias et al., 2022) reveal the features of the tumor cell population responsible for CRC recurrence, and have the potential to change the clinical management of CRC patients at risk of disease relapse.In addition, we have discovered that lack of optimal stem cell growth conditions in colorectal cancer organoids specifies a quiescent Lgr5+ cancer stem cell population that is responsible for relapse after chemotherapy. Quiescent Lgr5+ cancer stem cells can be identified by the expression of the gene Mex3a, are largely insensitive to chemotherapy, and regenerate the CRC organoid culture after treatment. In mouse models of metastatic latency, Mex3a+ cells contribute marginally to metastatic outgrowth. However, after chemotherapy treatment, Mex3a+ cells produce large cell clones that regenerate metastatic disease. Using lineage-tracing analysis combined with single cell profiling, we have shown that drug-tolerant persister Mex3a+ cells downregulate the WNT/Lgr5+ stem cell program immediately after chemotherapy and adopt a transient regenerative state reminiscent of that of fetal intestinal progenitors and revival gut stem cells. Mex3a KO tumours on the other hand, are unable to transition to the regenerative state, and instead differentiate towards a secretory phenotype. These findings reveal that adaptation of cancer stem cells to suboptimal niche environments protects them from drug treatment and identify a candidate cell of origin of relapse after chemotherapy in CRC. These findings have been published in the journal Nature Cancer (Alvarez-Varela et al., 2022) Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) 1.- The human-like pre-clinical model of advanced CRC that undergoes metastatic relapse after surgical resection of the primary tumour. A video illustrating the methodological/technical aspects was included in the publication, and a specific methodological publication for the journal Nature Protocols is being prepared. This model will allow further fundamental research as well as its use to assess therapies aiming to prevent disease relapse.2.- Single cell RNA sequencing datasets covering cell state dynamics of metastatic outgrowth (from micrometastases, to small metastases, to large metastases, in addition to their comparison with datasets from Primary Tumours). These important datasets have been deposited in public repositories so that the field can take advantage of the data generated during the execution of this proposal. Count matrices for single-cell RNA-seq experiments have been deposited at ArrayExpress under accession number E-MTAB-11284 (10x AKTP primary tumours), E-MTAB-11302 (Smart-seq metastatic progression) and E-MTAB-11981 (Smart-seq AKP micrometastases). Additional metadata and processed data files, including UMAP embeddings and gene signature scores, are available at Synapse (syn35000645).3.- Datasets covering RNA-seq data and microarray expression data that support the findings related to the discovery of Mex3a persister cells have been deposited at GEO under accession numbers GSE163171, GSE187650, GSE163035 and GSE187512. scRNA-seq experiments were deposited in ArrayExpress under accession numbers E-MTAB-11145 and E-MTAB-11146. High Relapse Cells (HRCs), responsible for disease relapse after surgery, lodged in the Liver.
