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Development of New Therapeutic Combinations for Ovarian Cancer Using Genetically Defined, Syngeneic Organoid Platform

ABSTRACT: The paucity of genetically informed, immune-competent tumor models impedes evaluation of conventional, targeted, and immune therapies. By engineering mouse fallopian tube (FT) organoids using lentiviral gene transduction and/or CRISPR/Cas9 mutagenesis, we generated multiple high grade serous ovarian carcinoma (HGSOC) models exhibiting combinations of mutations seen in patients. Detailed analysis of homologous recombination (HR)-proficient (Tp53-/-;Ccne1OE;Akt2OE ;KrasOE), HR-deficient (Tp53-/-;Brca1-/-;MycOE ) and unclassified (Tp53-/-;Pten-/-;Nf1-/-) examples revealed differences in in vitro properties and tumorigenicity/metastasis. Tumorigenic organoids had differential sensitivity to HGSOC chemotherapeutics and evoked distinct immune microenvironments. These findings enabled development of a chemotherapy/immunotherapy regimen that yielded durable, T-cell dependent responses in Tp53-/-;Ccne1OE;Akt2OE;Kras HGSOC; by contrast, Tp53-/-;Pten-/-;Nf1-/- tumors failed to respond. Genotype-informed, syngeneic organoid models could provide an improved platform for rapid evaluation of tumor biology and therapeutics.

ORGANISM(S): Mus musculus

PROVIDER: GSE147276 | GEO | 2020/11/08

REPOSITORIES: GEO

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Project description:High-grade serous ovarian cancer (HGSOC) creates a unique clinical challenge due to late detection, limited therapeutic options and wide-spread resistance to chemotherapy, all of which lead to very low survival rates. It is now widely recognized that this cancer emerges as metastasis from the fallopian tube and the lack of suitable in vitro disease models poses a major obstacle for efforts to improve our understanding of this deadly disease. We used organoids of healthy human FT epithelium as a model for stepwise genetic modification towards malignancy, using shRNA-mediated knockdown (KD) of three major HGSOC driver genes: p53, PTEN and Retinoblastoma (Rb). These pathways are known to be disrupted in the vast majority of HGSOC patients (>95%, 45% and 67% of cases, respectively as reported by TCGA consortium). Unexpectedly, combined loss of function of the three tumor suppressor proteins, p53, PTEN and Rb, led to a growth arrest and loss of stemness if organoids are cultured in medium optimized for healthy FT epithelium, which maintains high Wnt signaling and BMP pathway inhibition. This designates a requirement for alterations in the exogenous signaling cues to facilitate transformation. Indeed, we show that successful expansion of patient-derived HGSOC organoids from solid tumor deposits depends on the absence of exogenous Wnt3A in the medium. Using a combinatorial screening approach of different medium compositions, we have established 15 organoid lines, which closely resemble the parental tumor tissue and express hallmarks of HGSOC phenotype as revealed by histology and global gene expression profiles. Strikingly, the medium optimized for ovarian cancer organoids also rescued the growth of the modified organoid lines, showing that changes within the niche environment are required to promote stemness upon depletion of p53/PTEN/RB. Likewise, surface expression of the stemness marker CD133 was strongly increased in triple KD organoids in the absence of Wnt3A/Rspo1. Complementary to this finding, the addition of Wnt agonists to cancer organoids led to rapid growth arrest and a sharp drop in stemness, as evidenced by cell viability assay and the number of CD133 positive cells, respectively. Taken together, our data reveal that critical changes in the paracrine environment constitute early events during HGSOC development.

Project description:High-grade serous ovarian cancer (HGSOC) creates a unique clinical challenge due to late detection, limited therapeutic options and wide-spread resistance to chemotherapy, all of which lead to very low survival rates. It is now widely recognized that this cancer emerges as metastasis from the fallopian tube and the lack of suitable in vitro disease models poses a major obstacle for efforts to improve our understanding of this deadly disease. We used organoids of healthy human FT epithelium as a model for stepwise genetic modification towards malignancy, using shRNA-mediated knockdown (KD) of three major HGSOC driver genes: P53, PTEN and Retinoblastoma (RB). Moreover, we successfully established patient-derived HGSOC organoids from solid tumor deposits. After combinatorial screening approach of different media compositions we have established 15 organoid lines which depend on the absence of exogenous Wnt3a in the medium. We could show that those cultures closely resemble the parental tumor tissue and express hallmarks of HGSOC phenotype as revealed by histology and global gene expression profiles of the matching organoid and tissue samples. Expression and sequencing analysis revealed that all samples were characterized by p53 mutation and/or aberrant p53 expression. Interestingly, the addition of Wnt agonists to cancer organoids leads to rapid growth arrest and a sharp drop in stemness, as proven by cell viability assay and the number of CD133 positive cells, respectively. Complementary to this finding, we can show that the medium optimized for ovarian cancer organoids also improves the growth of the modified organoid lines, showing that changes within the niche environment are required to promote stemness upon depletion of P53/PTEN/RB. Taken together, our data reveal that critical changes in the paracrine environment are likely to represent early events during HGSOC development and provide a starting point to further analyze how specific mutations and paracrine signals influence pathways of stemness regulation and thus drive malignant transformation in ovarian carcinogenesis.

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Development of New Therapeutic Combinations for Ovarian Cancer Using Genetically Defined, Syngeneic Organoid Platform

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