Patient Derived Organoids Confirm That PI3K/AKT Signalling Is an Escape Pathway for Radioresistance and a Target for Therapy in Rectal Cancer

Abstract

ObjectivesPartial or total resistance to preoperative chemoradiotherapy occurs in more than half of locally advanced rectal cancer patients. Several novel or repurposed drugs have been trialled to improve cancer cell sensitivity to radiotherapy, with limited success. We aimed to understand the mechanisms of resistance to chemoradiotherapy in rectal cancer using patient derived organoid models.DesignTo understand the mechanisms underlying this resistance, we compared the pre-treatment transcriptomes of patient-derived organoids (PDO) with measured radiotherapy sensitivity to identify biological pathways involved in radiation resistance coupled with single cell sequencing, genome wide CRISPR-Cas9 and targeted drug screens.ResultsRNA sequencing enrichment analysis revealed upregulation of PI3K/AKT/mTOR and epithelial mesenchymal transition pathway genes in radioresistant PDOs. Single-cell sequencing of pre & post-irradiation PDOs showed mTORC1 and PI3K/AKT upregulation, which was confirmed by a genome-wide CRSIPR-Cas9 knockout screen using irradiated colorectal cancer (CRC) cell lines. We then tested the efficiency of dual PI3K/mTOR inhibitors in improving cancer cell sensitivity to radiotherapy. After irradiation, significant AKT phosphorylation was detected (p=0.027) which was abrogated with dual PI3K/mTOR inhibitors and lead to significant radiosensitisation of the HCT116 cell line and radiation resistant PDO lines.ConclusionsThe PI3K/AKT/mTOR pathway upregulation contributes to radioresistance and its targeted pharmacological inhibition leads to significant radiosensitisation in CRC organoids, making it a potential target for clinical trials.

Novel radiation and targeted therapy combinations for improving rectal cancer outcomes

“…Similarly, dactolisib (PI3K/mTOR inhibitor) and 5 fractions of 5 Gy in the same two PDO lines significantly reduced viability compared with drug alone ( P = 0.015, P = 0.002) (Ref. 62). An additional study observed that protein expression of phosphorylated Akt at serine 473 was elevated in rectal biopsies of patients who had poor response to CRT ( n = 50) (Ref.…”

“…In two PDO lines apitolisib (PI3K/mTOR inhibitor) combined with 5 fractions of 5 Gy significantly reduced viability when compared with drug alone ( P < 0.001) (Ref. 62). Similarly, dactolisib (PI3K/mTOR inhibitor) and 5 fractions of 5 Gy in the same two PDO lines significantly reduced viability compared with drug alone ( P = 0.015, P = 0.002) (Ref.…”

“…Radio-resistant HCT116 cell lines and PDOs’ dual inhibition of PI3K/mTOR were sensitised to radiation (Ref. 62). In two PDO lines apitolisib (PI3K/mTOR inhibitor) combined with 5 fractions of 5 Gy significantly reduced viability when compared with drug alone ( P < 0.001) (Ref.…”

PIM1 controls GBP1 activity to limit self-damage and to guard against pathogen infection

“…Patient-derived colorectal tumor organoids (Organoid 389), originally derived from a T3N1 poorly differentiated, mismatch repair–proficient adenocarcinoma of the sigmoid colon from a male subject ( 52 ), were grown and maintained in 50 μl of Matrigel domes (CLS356231, Corning) with 500 μl of human IntestiCult medium (06010, StemCell) in 24-well plates at 37°C and 5% CO 2 and passaged weekly.…”

Exploring New Dimensions of Tumor Heterogeneity: The Application of Single Cell Analysis to Organoid‐Based 3D In Vitro Models

“…For example, Wanigasooriya et al used clustering and pathway analysis of scRNA-seq data to explore the effect of radiation treatment on rectal cancer PDOs and identified the enrichment of stem cell marker-expressing cells and the upregulation of DNA repair and mTOR signaling pathways following radiation treatment. [197] Krieger et al used scRNA-seq to identify a differentiation trajectory for the subpopulations of cells present in PDAC PDOs, which showed that PDO growth was likely sustained by a large pool of cycling cells that differentiated to non-proliferating secretory cells. They also found that this differentiation trajectory was preserved between primary and metastatic sites, suggesting metastatic lesions share critical and potentially targetable aspects of the primary tumor.…”