Protocol for generation of and high-throughput drug testing with patient-derived colorectal cancer organoids.

Drug sensitivity testing of patient-derived tumor organoids (PDTOs) is a promising tool for personalizing cancer treatment. Here, we present a protocol for generation of and high-throughput drug testing with PDTOs. We describe detailed steps for PDTO establishment from colorectal cancer tissues, preparation of PDTOs for high-throughput drug testing, and quantification of drug testing results using image analysis. This protocol provides a standardized workflow for PDTO testing of standard-of-care therapies, along with exploring the activity of new agents, for translational research. For complete details on the use and execution of this protocol, please refer to Tan et al.1.

BCL-XL inhibitors enhance the apoptotic efficacy of BRAF inhibitors in BRAFV600E colorectal cancer.

Metastatic BRAFV600E colorectal cancer (CRC) carries an extremely poor prognosis and is in urgent need of effective new treatments. While the BRAFV600E inhibitor encorafenib in combination with the EGFR inhibitor cetuximab (Enc+Cet) was recently approved for this indication, overall survival is only increased by 3.6 months and objective responses are observed in only 20% of patients. We have found that a limitation of Enc+Cet treatment is the failure to efficiently induce apoptosis in BRAFV600E CRCs, despite inducing expression of the pro-apoptotic protein BIM and repressing expression of the pro-survival protein MCL-1. Here, we show that BRAFV600E CRCs express high basal levels of the pro-survival proteins MCL-1 and BCL-XL, and that combining encorafenib with a BCL-XL inhibitor significantly enhances apoptosis in BRAFV600E CRC cell lines. This effect was partially dependent on the induction of BIM, as BIM deletion markedly attenuated BRAF plus BCL-XL inhibitor-induced apoptosis. As thrombocytopenia is an established on-target toxicity of BCL-XL inhibition, we also examined the effect of combining encorafenib with the BCL-XL -targeting PROTAC DT2216, and the novel BCL-2/BCL-XL inhibitor dendrimer conjugate AZD0466. Combining encorafenib with DT2216 significantly increased apoptosis induction in vitro, while combining encorafenib with AZD0466 was well tolerated in mice and further reduced growth of BRAFV600E CRC xenografts compared to either agent alone. Collectively, these findings demonstrate that combined BRAF and BCL-XL inhibition significantly enhances apoptosis in pre-clinical models of BRAFV600E CRC and is a combination regimen worthy of clinical investigation to improve outcomes for these patients.

Loss-of-Function but Not Gain-of-Function Properties of Mutant TP53 Are Critical for the Proliferation, Survival, and Metastasis of a Broad Range of Cancer Cells.

Mutations in the tumor suppressor TP53 cause cancer and impart poor chemotherapeutic responses, reportedly through loss-of-function, dominant-negative effects and gain-of-function (GOF) activities. The relative contributions of these attributes is unknown. We found that removal of 12 different TP53 mutants with reported GOFs by CRISPR/Cas9 did not impact proliferation and response to chemotherapeutics of 15 human cancer cell lines and colon cancer-derived organoids in culture. Moreover, removal of mutant TP53/TRP53 did not impair growth or metastasis of human cancers in immune-deficient mice or growth of murine cancers in immune-competent mice. DepMap mining revealed that removal of 158 different TP53 mutants had no impact on the growth of 391 human cancer cell lines. In contrast, CRISPR-mediated restoration of wild-type TP53 extinguished the growth of human cancer cells in vitro. These findings demonstrate that LOF but not GOF effects of mutant TP53/TRP53 are critical to sustain expansion of many tumor types. SIGNIFICANCE: This study provides evidence that removal of mutant TP53, thereby deleting its reported GOF activities, does not impact the survival, proliferation, metastasis, or chemotherapy responses of cancer cells. Thus, approaches that abrogate expression of mutant TP53 or target its reported GOF activities are unlikely to exert therapeutic impact in cancer. See related commentary by Lane, p. 211 . This article is featured in Selected Articles from This Issue, p. 201.

Unified framework for patient-derived, tumor-organoid-based predictive testing of standard-of-care therapies in metastatic colorectal cancer.

Predictive drug testing of patient-derived tumor organoids (PDTOs) holds promise for personalizing treatment of metastatic colorectal cancer (mCRC), but prospective data are limited to chemotherapy regimens with conflicting results. We describe a unified framework for PDTO-based predictive testing across standard-of-care chemotherapy and biologic and targeted therapy options. In an Australian community cohort, PDTO predictions based on treatment-naive patients (n = 56) and response rates from first-line mCRC clinical trials achieve 83% accuracy for forecasting responses in patients receiving palliative treatments (18 patients, 29 treatments). Similar assay accuracy is achieved in a prospective study of third-line or later mCRC treatment, AGITG FORECAST-1 (n = 30 patients). "Resistant" predictions are associated with inferior progression-free survival; misclassification rates are similar by regimen. Liver metastases are the optimal site for sampling, with testing achievable within 7 weeks for 68.8% cases. Our findings indicate that PDTO drug panel testing can provide predictive information for multifarious standard-of-care therapies for mCRC.

Oncomicrobial Community Profiling Identifies Clinicomolecular and Prognostic Subtypes of Colorectal Cancer.

BACKGROUND & AIMS: Dysbiosis of gut microbiota is linked to the development of colorectal cancer (CRC). However, microbiota-based stratification of CRC tissue and how this relates to clinicomolecular characteristics and prognosis remains to be clarified. METHODS: Tumor and normal mucosa from 423 patients with stage I to IV CRC were profiled by bacterial 16S rRNA gene sequencing. Tumors were characterized for microsatellite instability (MSI), CpG island methylator phenotype (CIMP), APC, BRAF, KRAS, PIK3CA, FBXW7, SMAD4, and TP53 mutations, subsets for chromosome instability (CIN), mutation signatures, and consensus molecular subtypes (CMS). Microbial clusters were validated in an independent cohort of 293 stage II/III tumors. RESULTS: Tumors reproducibly stratified into 3 oncomicrobial community subtypes (OCSs) with distinguishing features: OCS1 (Fusobacterium/oral pathogens, proteolytic, 21%), right-sided, high-grade, MSI-high, CIMP-positive, CMS1, BRAF V600E, and FBXW7 mutated; OCS2 (Firmicutes/Bacteroidetes, saccharolytic, 44%), and OCS3 (Escherichia/Pseudescherichia/Shigella, fatty acid ß-oxidation, 35%) both left-sided and exhibiting CIN. OCS1 was associated with MSI-related mutation signatures (SBS15, SBS20, ID2, and ID7) and OCS2 and OCS3 with SBS18 related to damage by reactive oxygen species. Among stage II/III patients, OCS1 and OCS3 both had poorer overall survival compared with OCS2 for microsatellite stable tumors (multivariate hazard ratio [HR], 1.85; 95% confidence interval [CI], 1.15-2.99; P = .012; and HR, 1.52; 95% CI 1.01-2.29; P = .044, respectively) and left-sided tumors (multivariate HR, 2.66; 95% CI, 1.45-4.86; P = .002; and HR, 1.76; 95% CI, 1.03-3.02; P = .039, respectively). CONCLUSIONS: OCS classification stratified CRCs into 3 distinct subgroups with different clinicomolecular features and outcomes. Our findings provide a framework for a microbiota-based stratification of CRC to refine prognostication and to inform the development of microbiota-targeted interventions.

PI3Kα Translocation Mediates Nuclear PtdIns(3,4,5)P3 Effector Signaling in Colorectal Cancer.

The canonical view of PI3Kα signaling describes phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) generation and activation of downstream effectors at the plasma membrane or at microtubule-bound endosomes. Here, we show that colorectal cancer (CRC) cell lines exhibit a diverse plasma membrane-nuclear distribution of PI3Kα, controlling corresponding levels of subcellular PtdIns(3,4,5)P3 pools. PI3Kα nuclear translocation was mediated by the importin ß-dependent nuclear import pathway. By PtdIns(3,4,5)P3 affinity capture mass spectrometry done in the presence of SDS on CRC cell lines with PI3Kα nuclear localization, we identified 867 potential nuclear PtdIns(3,4,5)P3 effector proteins. Nuclear PtdIns(3,4,5)P3 interactome proteins were characterized by noncanonical PtdIns(3,4,5)P3-binding domains and showed overrepresentation for nuclear membrane, nucleolus, and nuclear speckles. The nuclear PtdIns(3,4,5)P3 interactome was enriched for proteins related to RNA metabolism, with splicing reporter assays and SC-35 foci staining suggesting a role of epidermal growth factor-stimulated nuclear PI3Kα signaling in modulating pre-mRNA splicing. In patient tumors, nuclear p110α staining was associated with lower T stage and mucinous histology. These results indicate that PI3Kα translocation mediates nuclear PtdIns(3,4,5)P3 effector signaling in human CRC, modulating signaling responses.

The anti-cancer agent APR-246 can activate several programmed cell death processes to kill malignant cells.

Mutant TP53 proteins are thought to drive the development and sustained expansion of cancers at least in part through the loss of the wild-type (wt) TP53 tumour suppressive functions. Therefore, compounds that can restore wt TP53 functions in mutant TP53 proteins are expected to inhibit the expansion of tumours expressing mutant TP53. APR-246 has been reported to exert such effects in malignant cells and is currently undergoing clinical trials in several cancer types. However, there is evidence that APR-246 may also kill malignant cells that do not express mutant TP53. To support the clinical development of APR-246 it is important to understand its mechanism(s) of action. By establishing isogenic background tumour cell lines with different TP53/TRP53 states, we found that APR-246 can kill malignant cells irrespective of their TP53/TRP53 status. Accordingly, RNAseq analysis revealed that treatment with APR-246 induces expression of the same gene set in Eµ-Myc mouse lymphoma cells of all four possible TRP53 states, wt, wt alongside mutant, knockout and knockout alongside mutant. We found that depending on the type of cancer cell and the concentration of APR-246 used, this compound can kill malignant cells through induction of various programmed cell death pathways, including apoptosis, necroptosis and ferroptosis. The sensitivity of non-transformed cells to APR-246 also depended on the cell type. These findings reveal that the clinical testing of APR-246 should not be limited to cancers expressing mutant TP53 but expanded to cancers that express wt TP53 or are TP53-deficient.

Evaluating Multiple Next-Generation Sequencing-Derived Tumor Features to Accurately Predict DNA Mismatch Repair Status.

Identifying tumor DNA mismatch repair deficiency (dMMR) is important for precision medicine. Tumor features, individually and in combination, derived from whole-exome sequenced (WES) colorectal cancers (CRCs) and panel-sequenced CRCs, endometrial cancers (ECs), and sebaceous skin tumors (SSTs) were assessed for their accuracy in detecting dMMR. CRCs (n = 300) with WES, where mismatch repair status was determined by immunohistochemistry, were assessed for microsatellite instability (MSMuTect, MANTIS, MSIseq, and MSISensor), Catalogue of Somatic Mutations in Cancer tumor mutational signatures, and somatic mutation counts. A 10-fold cross-validation approach (100 repeats) evaluated the dMMR prediction accuracy for i) individual features, ii) Lasso statistical model, and iii) an additive feature combination approach. Panel-sequenced tumors (29 CRCs, 22 ECs, and 20 SSTs) were assessed for the top performing dMMR predicting features/models using these three approaches. For WES CRCs, 10 features provided >80% dMMR prediction accuracy, with MSMuTect, MSIseq, and MANTIS achieving ≥99% accuracy. The Lasso model achieved 98.3% accuracy. The additive feature approach, with three or more of six of MSMuTect, MANTIS, MSIseq, MSISensor, insertion-deletion count, or tumor mutational signature small insertion/deletion 2 + small insertion/deletion 7 achieved 99.7% accuracy. For the panel-sequenced tumors, the additive feature combination approach of three or more of six achieved accuracies of 100%, 95.5%, and 100% for CRCs, ECs, and SSTs, respectively. The microsatellite instability calling tools performed well in WES CRCs; however, an approach combining tumor features may improve dMMR prediction in both WES and panel-sequenced data across tissue types.

Genotype-Tailored ERK/MAPK Pathway and HDAC Inhibition Rewires the Apoptotic Rheostat to Trigger Colorectal Cancer Cell Death.

The EGFR/RAS/MEK/ERK signaling pathway (ERK/MAPK) is hyperactivated in most colorectal cancers. A current limitation of inhibitors of this pathway is that they primarily induce cytostatic effects in colorectal cancer cells. Nevertheless, these drugs do induce expression of proapoptotic factors, suggesting they may prime colorectal cancer cells to undergo apoptosis. As histone deacetylase inhibitors (HDACis) induce expression of multiple proapoptotic proteins, we examined whether they could synergize with ERK/MAPK inhibitors to trigger colorectal cancer cell apoptosis. Combined MEK/ERK and HDAC inhibition synergistically induced apoptosis in colorectal cancer cell lines and patient-derived tumor organoids in vitro, and attenuated Apc-initiated adenoma formation in vivo. Mechanistically, combined MAPK/HDAC inhibition enhanced expression of the BH3-only proapoptotic proteins BIM and BMF, and their knockdown significantly attenuated MAPK/HDAC inhibitor-induced apoptosis. Importantly, we demonstrate that the paradigm of combined MAPK/HDAC inhibitor treatment to induce apoptosis can be tailored to specific MAPK genotypes in colorectal cancers, by combining an HDAC inhibitor with either an EGFR, KRASG12C or BRAFV600 inhibitor in KRAS/BRAFWT; KRASG12C, BRAFV600E colorectal cancer cell lines, respectively. These findings identify a series of ERK/MAPK genotype-tailored treatment strategies that can readily undergo clinical testing for the treatment of colorectal cancer.

Reprogrammed Lipid Metabolism and the Lipid-Associated Hallmarks of Colorectal Cancer.

Lipids have diverse structures, with multifarious regulatory functions in membrane homeostasis and bioenergetic metabolism, in mediating functional protein-lipid and protein-protein interactions, as in cell signalling and proliferation. An increasing body of evidence supports the notion that aberrant lipid metabolism involving remodelling of cellular membrane structure and changes in energy homeostasis and signalling within cancer-associated pathways play a pivotal role in the onset, progression, and maintenance of colorectal cancer (CRC) and their tumorigenic properties. Recent advances in analytical lipidome analysis technologies have enabled the comprehensive identification and structural characterization of lipids and, consequently, our understanding of the role they play in tumour progression. However, despite progress in our understanding of cancer cell metabolism and lipidomics, the key lipid-associated changes in CRC have yet not been explicitly associated with the well-established 'hallmarks of cancer' defined by Hanahan and Weinberg. In this review, we summarize recent findings that highlight the role of reprogrammed lipid metabolism in CRC and use this growing body of evidence to propose eight lipid metabolism-associated hallmarks of colorectal cancer, and to emphasize their importance and linkages to the established cancer hallmarks.

Low-viscosity Matrix Suspension Culture for Human Colorectal Epithelial Organoids and Tumoroids.

Three-dimensional culture of human normal colorectal epithelium and cancer tissue as organoids and tumoroids has transformed the study of diseases of the large intestine. A widely used strategy for generating patient-derived colorectal organoids and tumoroids involves embedding cells in domes of extracellular matrix (ECM). Despite its success, dome culture is not ideal for scalable expansion, experimentation, and high-throughput screening applications. Our group has developed a protocol for growing patient-derived colorectal organoids and tumoroids in low-viscosity matrix (LVM) suspension culture. Instead of embedding colonic crypts or tumor fragments in solid ECM, these are grown suspended in medium containing only a low percentage of ECM. Compared with dome cultures, LVM suspension culture reduces the labor and cost of establishing and passaging organoids and tumoroids, enables rapid expansion, and is readily adaptable for high-throughput screening. Graphic abstract: Generation of organoids and tumoroids from human large intestine using LVM suspension culture (Created with BioRender.com).

Beyond standard data collection - the promise and potential of BRAIN (Brain tumour Registry Australia INnovation and translation registry).

BACKGROUND: Real-world data (RWD) is increasingly being embraced as an invaluable source of information to address clinical and policy-relevant questions that are unlikely to ever be answered by clinical trials. However, the largely unrealised potential of RWD is the value to be gained by supporting prospective studies and translational research. Here we describe the design and implementation of an Australian brain cancer registry, BRAIN, which is pursuing these opportunities. METHODS: BRAIN was designed by a panel of clinicians in conjunction with BIOGRID to capture comprehensive clinical data on patients diagnosed with brain tumours from diagnosis through treatment to recurrence or death. Extensive internal and external testing was undertaken, followed by implementation at multiple sites across Victoria and Tasmania. RESULTS: Between February 2021 and December 2021, a total of 350 new patients from 10 sites, including one private and two regional, were entered into BRAIN. Additionally, BRAIN supports the world's first registry trial in neuro-oncology, EX-TEM, addressing the optimal duration of post-radiation temozolomide; and BioBRAIN, a dedicated brain tumour translational program providing a pipeline for biospecimen collection matched with linked clinical data. CONCLUSIONS: Here we report on the first data collection effort in brain tumours for Australia, which we believe to be unique worldwide given the number of sites and patients involved and the extent to which the registry resource is being leveraged to support clinical and translational research. Further directions such as passive data flow and data linkages, use of artificial intelligence and inclusion of patient-entered data are being explored.

Epithelial de-differentiation triggered by co-ordinate epigenetic inactivation of the EHF and CDX1 transcription factors drives colorectal cancer progression.

Colorectal cancers (CRCs) often display histological features indicative of aberrant differentiation but the molecular underpinnings of this trait and whether it directly drives disease progression is unclear. Here, we identify co-ordinate epigenetic inactivation of two epithelial-specific transcription factors, EHF and CDX1, as a mechanism driving differentiation loss in CRCs. Re-expression of EHF and CDX1 in poorly-differentiated CRC cells induced extensive chromatin remodelling, transcriptional re-programming, and differentiation along the enterocytic lineage, leading to reduced growth and metastasis. Strikingly, EHF and CDX1 were also able to reprogramme non-colonic epithelial cells to express colonic differentiation markers. By contrast, inactivation of EHF and CDX1 in well-differentiated CRC cells triggered tumour de-differentiation. Mechanistically, we demonstrate that EHF physically interacts with CDX1 via its PNT domain, and that these transcription factors co-operatively drive transcription of the colonic differentiation marker, VIL1. Compound genetic deletion of Ehf and Cdx1 in the mouse colon disrupted normal colonic differentiation and significantly enhanced colorectal tumour progression. These findings thus reveal a novel mechanism driving epithelial de-differentiation and tumour progression in CRC.

Cancer Tissue Classification Using Supervised Machine Learning Applied to MALDI Mass Spectrometry Imaging.

Matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) can determine the spatial distribution of analytes such as protein distributions in a tissue section according to their mass-to-charge ratio. Here, we explored the clinical potential of machine learning (ML) applied to MALDI MSI data for cancer diagnostic classification using tissue microarrays (TMAs) on 302 colorectal (CRC) and 257 endometrial cancer (EC)) patients. ML based on deep neural networks discriminated colorectal tumour from normal tissue with an overall accuracy of 98% in balanced cross-validation (98.2% sensitivity and 98.6% specificity). Moreover, our machine learning approach predicted the presence of lymph node metastasis (LNM) for primary tumours of EC with an accuracy of 80% (90% sensitivity and 69% specificity). Our results demonstrate the capability of MALDI MSI for complementing classic histopathological examination for cancer diagnostic applications.

Low-viscosity matrix suspension culture enables scalable analysis of patient-derived organoids and tumoroids from the large intestine.

Cell embedment into a solid support matrix is considered essential for the culture of intestinal epithelial organoids and tumoroids, but this technique presents challenges that impede scalable culture expansion, experimental manipulation, high-throughput screening and diagnostic applications. We have developed a low-viscosity matrix (LVM) suspension culture method that enables efficient establishment and propagation of organoids and tumoroids from the human large intestine. Organoids and tumoroids cultured in LVM suspension recapitulate the morphological development observed in solid matrices, with tumoroids reflecting the histological features and genetic heterogeneity of primary colorectal cancers. We demonstrate the utility of LVM suspension culture for organoid and tumoroid bioreactor applications and biobanking, as well as tumoroid high-throughput drug sensitivity testing. These methods provide opportunities for the study and use of patient-derived organoids and tumoroids from the large intestine.

EHF is essential for epidermal and colonic epithelial homeostasis, and suppresses Apc-initiated colonic tumorigenesis.

Ets homologous factor (EHF) is a member of the epithelial-specific Ets (ESE) family of transcription factors. To investigate its role in development and epithelial homeostasis, we generated a series of novel mouse strains in which the Ets DNA-binding domain of Ehf was deleted in all tissues (Ehf-/-) or specifically in the gut epithelium. Ehf-/- mice were born at the expected Mendelian ratio, but showed reduced body weight gain, and developed a series of pathologies requiring most Ehf-/- mice to reach an ethical endpoint before reaching 1 year of age. These included papillomas in the facial skin, abscesses in the preputial glands (males) or vulvae (females), and corneal ulcers. Ehf-/-mice also displayed increased susceptibility to experimentally induced colitis, which was confirmed in intestinal-specific Ehf knockout mice. Gut-specific Ehf deletion also impaired goblet cell differentiation, induced extensive transcriptional reprogramming in the colonic epithelium and enhanced Apc-initiated adenoma development. The Ets DNA-binding domain of EHF is therefore essential for postnatal homeostasis of the epidermis and colonic epithelium, and its loss promotes colonic tumour development.

Genomic Signature of Oral Squamous Cell Carcinomas from Non-Smoking Non-Drinking Patients.

Molecular alterations in 176 patients with oral squamous cell carcinomas (OSCC) were evaluated to delineate differences in non-smoking non-drinking (NSND) patients. Somatic mutations and DNA copy number variations (CNVs) in a 68-gene panel and human papilloma virus (HPV) status were interrogated using targeted next-generation sequencing. In the entire cohort, TP53 (60%) and CDKN2A (24%) were most frequently mutated, and the most common CNVs were EGFR amplifications (9%) and deletions of BRCA2 (5%) and CDKN2A (4%). Significant associations were found for TP53 mutation and nodal disease, lymphovascular invasion and extracapsular spread, CDKN2A mutation or deletion with advanced tumour stage, and EGFR amplification with perineural invasion and extracapsular spread. PIK3CA mutation, CDKN2A deletion, and EGFR amplification were associated with worse survival in univariate analyses (p < 0.05 for all comparisons). There were 59 NSND patients who tended to be female and older than patients who smoke and/or drink, and showed enrichment of CDKN2A mutations, EGFR amplifications, and BRCA2 deletions (p < 0.05 for all comparisons), with a younger subset showing higher mutation burden. HPV was detected in three OSCC patients and not associated with smoking and drinking habits. NSND OSCC exhibits distinct genomic profiles and further exploration to elucidate the molecular aetiology in these patients is warranted.

Evaluating the utility of tumour mutational signatures for identifying hereditary colorectal cancer and polyposis syndrome carriers.

OBJECTIVE: Germline pathogenic variants (PVs) in the DNA mismatch repair (MMR) genes and in the base excision repair gene MUTYH underlie hereditary colorectal cancer (CRC) and polyposis syndromes. We evaluated the robustness and discriminatory potential of tumour mutational signatures in CRCs for identifying germline PV carriers. DESIGN: Whole-exome sequencing of formalin-fixed paraffin-embedded (FFPE) CRC tissue was performed on 33 MMR germline PV carriers, 12 biallelic MUTYH germline PV carriers, 25 sporadic MLH1 methylated MMR-deficient CRCs (MMRd controls) and 160 sporadic MMR-proficient CRCs (MMRp controls) and included 498 TCGA CRC tumours. COSMIC V3 single base substitution (SBS) and indel (ID) mutational signatures were assessed for their ability to differentiate CRCs that developed in carriers from non-carriers. RESULTS: The combination of mutational signatures SBS18 and SBS36 contributing >30% of a CRC's signature profile was able to discriminate biallelic MUTYH carriers from all other non-carrier control CRCs with 100% accuracy (area under the curve (AUC) 1.0). SBS18 and SBS36 were associated with specific MUTYH variants p.Gly396Asp (p=0.025) and p.Tyr179Cys (p=5×10-5), respectively. The combination of ID2 and ID7 could discriminate the 33 MMR PV carrier CRCs from the MMRp control CRCs (AUC 0.99); however, SBS and ID signatures, alone or in combination, could not provide complete discrimination (AUC 0.79) between CRCs from MMR PV carriers and sporadic MMRd controls. CONCLUSION: Assessment of SBS and ID signatures can discriminate CRCs from biallelic MUTYH carriers and MMR PV carriers from non-carriers with high accuracy, demonstrating utility as a potential diagnostic and variant classification tool.