Functional screening of amplification outlier oncogenes in organoid models of early tumorigenesis

Genomics; Organoid; Squamous cancerGenómica funcional; Organoide; Cáncer escamosoGenòmica funcional; Organoide; Càncer escamósSomatic copy number gains are pervasive across cancer types, yet their roles in oncogenesis are insufficiently evaluated. This inadequacy is partly due to copy gains spanning large chromosomal regions, obscuring causal loci. Here, we employed organoid modeling to evaluate candidate oncogenic loci identified via integrative computational analysis of extreme copy gains overlapping with extreme expression dysregulation in The Cancer Genome Atlas. Subsets of “outlier” candidates were contextually screened as tissue-specific cDNA lentiviral libraries within cognate esophagus, oral cavity, colon, stomach, pancreas, and lung organoids bearing initial oncogenic mutations. Iterative analysis nominated the kinase DYRK2 at 12q15 as an amplified head and neck squamous carcinoma oncogene in p53−/− oral mucosal organoids. Similarly, FGF3, amplified at 11q13 in 41% of esophageal squamous carcinomas, promoted p53−/− esophageal organoid growth reversible by small molecule and soluble receptor antagonism of FGFRs. Our studies establish organoid-based contextual screening of candidate genomic drivers, enabling functional evaluation during early tumorigenesis.This work was supported by the NCI Cancer Target Discovery and Development (CTD∧2) Network (U01CA217851, C.J.K and C.C.; U01CA176058, W.C.H.). Support was also provided by NIH K08DE027730 and D.R. discretionary funds to A.A.S., AEI RYC2019- 026576-I, “LaCaixa” Foundation LCF/PR/PR17/51120011 to J.A.S., and NIH U54CA224081, NIH U01CA199241, Emerson Collective, Ludwig Cancer Research, and Stand Up To Cancer to C.J.K. This manuscript is dedicated to the memories of Dr. Daniela Gerhard and Dr. Kenneth Scott

Preclinical Evaluation of Artesunate as an Antineoplastic Agent in Ovarian Cancer Treatment

BACKGROUND: Ovarian cancer is the deadliest gynecologic malignancy despite current first-line treatment with a platinum and taxane doublet. Artesunate has broad antineoplastic properties but has not been investigated in combination with carboplatin and paclitaxel for ovarian cancer treatment. METHODS: Standard cell culture technique with commercially available ovarian cancer cell lines were utilized in cell viability, DNA damage, and cell cycle progression assays to qualify and quantify artesunate treatment effects. Additionally, the sequence of administering artesunate in combination with paclitaxel and carboplatin was determined. The activity of artesunate was also assessed in 3D organoid models of primary ovarian cancer and RNAseq analysis was utilized to identify genes and the associated genetic pathways that were differentially regulated in artesunate resistant organoid models compared to organoids that were sensitive to artesunate. RESULTS: Artesunate treatment reduces cell viability in 2D and 3D ovarian cancer cell models. Clinically relevant concentrations of artesunate induce G1 arrest, but do not induce DNA damage. Pathways related to cell cycle progression, specifically G1/S transition, are upregulated in ovarian organoid models that are innately more resistant to artesunate compared to more sensitive models. Depending on the sequence of administration, the addition of artesunate to carboplatin and paclitaxel improves their effectiveness. CONCLUSIONS: Artesunate has preclinical activity in ovarian cancer that merits further investigation to treat ovarian cancer

Refining colorectal cancer classification and clinical stratification through a single-cell atlas

Background Colorectal cancer (CRC) consensus molecular subtypes (CMS) have different immunological, stromal cell, and clinicopathological characteristics. Single-cell characterization of CMS subtype tumor microenvironments is required to elucidate mechanisms of tumor and stroma cell contributions to pathogenesis which may advance subtype-specific therapeutic development. We interrogate racially diverse human CRC samples and analyze multiple independent external cohorts for a total of 487,829 single cells enabling high-resolution depiction of the cellular diversity and heterogeneity within the tumor and microenvironmental cells. Results Tumor cells recapitulate individual CMS subgroups yet exhibit significant intratumoral CMS heterogeneity. Both CMS1 microsatellite instability (MSI-H) CRCs and microsatellite stable (MSS) CRC demonstrate similar pathway activations at the tumor epithelial level. However, CD8+ cytotoxic T cell phenotype infiltration in MSI-H CRCs may explain why these tumors respond to immune checkpoint inhibitors. Cellular transcriptomic profiles in CRC exist in a tumor immune stromal continuum in contrast to discrete subtypes proposed by studies utilizing bulk transcriptomics. We note a dichotomy in tumor microenvironments across CMS subgroups exists by which patients with high cancer-associated fibroblasts (CAFs) and C1Q+TAM content exhibit poor outcomes, providing a higher level of personalization and precision than would distinct subtypes. Additionally, we discover CAF subtypes known to be associated with immunotherapy resistance. Conclusions Distinct CAFs and C1Q+ TAMs are sufficient to explain CMS predictive ability and a simpler signature based on these cellular phenotypes could stratify CRC patient prognosis with greater precision. Therapeutically targeting specific CAF subtypes and C1Q + TAMs may promote immunotherapy responses in CRC patient

Redefining tumor classification and clinical stratification through a colorectal cancer single-cell atlas

Colorectal cancer (CRC), a disease of high incidence and mortality, exhibits a large degree of inter- and intra-tumoral heterogeneity. The cellular etiology of this heterogeneity is poorly understood. Here, we generated and analyzed a single-cell transcriptome atlas of 49,859 CRC cells from 16 patients, validated with an additional 31,383 cells from an independent CRC patient cohort. We describe subclonal transcriptomic heterogeneity of CRC tumor epithelial cells, as well as discrete stromal populations of cancer-associated fibroblasts (CAFs). Within CRC CAFs, we identify the transcriptional signature of specific subtypes that significantly stratifies overall survival in more than 1,500 CRC patients with bulk transcriptomic data. We demonstrate that scRNA analysis of malignant, stromal, and immune cells exhibit a more complex picture than portrayed by bulk transcriptomic-based Consensus Molecular Subtypes (CMS) classification. By demonstrating an abundant degree of heterogeneity amongst these cell types, our work shows that CRC is best represented in a transcriptomic continuum crossing traditional classification systems boundaries. Overall, this CRC cell map provides a framework to re-evaluate CRC tumor biology with implications for clinical trial design and therapeutic development. Competing Interest Statement: The authors have declared no competing interest

Progenitor identification and SARS-CoV-2 infection in human distal lung organoids

The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Three-dimensional in vitro human distal lung culture systems would strongly facilitate investigation of pathologies including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. We generated long-term feeder-free, chemically defined culture of distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids exhibited AT1 transdifferentiation potential while basal cell organoids developed lumens lined by differentiated club and ciliated cells. Single cell analysis of basal organoid KRT5+ cells revealed a distinct ITGA6+ITGB4+ mitotic population whose proliferation further segregated to a TNFRSF12Ahi subfraction comprising ~10% of KRT5+ basal cells, residing in clusters within terminal bronchioles and exhibiting enriched clonogenic organoid growth activity. Distal lung organoids were created with apical-out polarity to display ACE2 on the exposed external surface, facilitating SARS-CoV-2 infection of AT2 and basal cultures and identifying club cells as a novel target population. This long-term, feeder-free organoid culture of human distal lung, coupled with single cell analysis, identifies unsuspected basal cell functional heterogeneity and establishes a facile in vitro organoid model for human distal lung infections including COVID-19-associated pneumonia

Update on International Cooperative Groups Studies in Thoracic Malignancies: The Emergence of Immunotherapy.

Cancer cooperative groups have historically played a critical role in the advancement of non-small-cell lung cancer therapy. Representatives from cooperative groups worldwide convene at the International Lung Cancer Congress annually. The International Lung Cancer Congress had its 17th anniversary in the summer of 2016. The present review highlights the thoracic malignancy studies discussed by presenters. The included studies are merely a sample of the trials of thoracic malignancies ongoing globally

Update on International Cooperative Groups Studies in Thoracic Malignancies: The Emergence of Immunotherapy.

Cancer cooperative groups have historically played a critical role in the advancement of non-small-cell lung cancer therapy. Representatives from cooperative groups worldwide convene at the International Lung Cancer Congress annually. The International Lung Cancer Congress had its 17th anniversary in the summer of 2016. The present review highlights the thoracic malignancy studies discussed by presenters. The included studies are merely a sample of the trials of thoracic malignancies ongoing globally

Zinc Protoporphyrin Regulates Cyclin D1 Expression Independent of Heme Oxygenase Inhibition*

Zinc protoporphyrin IX (ZnPP), an endogenous heme analogue that inhibits heme oxygenase (HO) activity, represses tumor growth. It can also translocate into the nucleus and up-regulate heme oxygenase 1 (HMOX1) gene expression. Here, we demonstrate that tumor cell proliferation was inhibited by ZnPP, whereas tin protoporphyrin (SnPP), another equally potent HO-1 inhibitor, had no effect. Microarray analysis on 128 tumorigenesis related genes showed that ZnPP suppressed genes involved in cell proliferation and angiogenesis. Among these genes, CYCLIN D1 (CCND1) was specifically inhibited as were its mRNA and protein levels. Additionally, ZnPP inhibited CCND1 promoter activity through an Sp1 and Egr1 overlapping binding site (S/E). We confirmed that ZnPP modulated the S/E site, at least partially by associating with Sp1 and Egr1 proteins rather than direct binding to DNA targets. Furthermore, administration of ZnPP significantly inhibited cyclin D1 expression and progression of a B-cell leukemia/lymphoma 1 tumor in mice by preferentially targeting tumor cells. These observations show HO independent effects of ZnPP on cyclin D1 expression and tumorigenesis