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Cited by 30Cancer Genomics and DiagnosticsPancreatic and Hepatic Oncology ResearchSingle-cell and spatial transcriptomics

Occult polyclonality of preclinical pancreatic cancer models drives in vitro evolution

Open access
Monberg, Maria E., Geiger, Heather, Lee, Jaewon J., Sharma, Roshan, Semaan, Alexander, Bernard, Vincent, Wong, Justin, Wang, Fang +8 more·2022·Nature Communications
DOI 10.1038/s41467-022-31376-3PMC PMC9233687

Published version of a preprint

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Abstract

Heterogeneity is a hallmark of cancer. The advent of single-cell technologies has helped uncover heterogeneity in a high-throughput manner in different cancers across varied contexts. Here we apply single-cell sequencing technologies to reveal inherent heterogeneity in assumptively monoclonal pancreatic cancer (PDAC) cell lines and patient-derived organoids (PDOs). Our findings reveal a high degree of both genomic and transcriptomic polyclonality in monolayer PDAC cell lines, custodial variation induced by growing apparently identical cell lines in different laboratories, and transcriptomic shifts in transitioning from 2D to 3D spheroid growth models. Our findings also call into question the validity of widely available immortalized, non-transformed pancreatic lines as contemporaneous “control” lines in experiments. We confirm these findings using a variety of independent assays, including but not limited to whole exome sequencing, single-cell copy number variation sequencing (scCNVseq), single-nuclei assay for transposase-accessible chromatin with sequencing, fluorescence in-situ hybridization, and single-cell RNA sequencing (scRNAseq). We map scRNA expression data to unique genomic clones identified by orthogonally-gathered scCNVseq data of these same PDAC cell lines. Further, while PDOs are known to reflect the cognate in vivo biology of the parental tumor, we identify transcriptomic shifts during ex vivo passage that might hamper their predictive abilities over time. The impact of these findings on rigor and reproducibility of experimental data generated using established preclinical PDAC models between and across laboratories is uncertain, but a matter of concern.

AI snapshot

Written from the paper’s full text.

TL;DR

Single-cell genomic and transcriptomic profiling of commonly used pancreatic cancer cell lines, “normal” controls, and patient-derived organoids reveals unexpected polyclonality, cross-laboratory divergence, and culture-induced transcriptomic/epigenetic shifts. The authors argue these occult differences undermine assumptions of clonality and reproducibility in preclinical PDAC models.

Key findings

  • Across six pancreatic cell lines and nine scRNA-seq samples (77,068 cells), the authors found substantial subclonal heterogeneity in assumptively monoclonal PDAC lines; seven samples also had scCNV-seq. HPDE “control” cells showed bi-allelic losses at 3p, 8p, and 9p, a chromosome 20q amplification (CN > 10 in a minor fraction of subclones) spanning AURKA, and AURKA transcript levels 2–4 fold higher than HPNE in at least 20% of cells.
  • HPNE cells were comparatively homogeneous by scCNV but had a 17q amplification including BRCA1, with at least 24% of HPNE cells highly expressing BRCA1 relative to HPDE.
  • Three MiaPaca2 strains from different sources showed transcriptomic divergence: MP2-A and MP2-C were entirely Moffitt “basal-like” (9,768 and 10,886 cells), while MP2-B was admixed (3,456 basal-like and 3,556 classical). Clonealign mapped scCNV clones to scRNA subpopulations, e.g., MP2-B subclone 1 had 1,096 cells and subclone 2 had 813 cells, with distinct oncogenic pathway enrichment.
  • Panc1 2D versus 3D spheroid growth showed epigenetic remodeling by snATAC-seq (9,785 nuclei in 2D, 7,453 in 3D), with Sp/Klf motif enrichment in spheroids and Fos/Jun motif enrichment in 2D. A PDO profiled at early and late passage, separated by 10 passages, developed two transcriptional fates and inferred chromosome 16p gain plus chromosome 19q loss in one late-passage fate.

Methods

  • Cultured PDAC lines (Panc-1, MiaPaCa2, HPAF-II, BxPC-3), immortalized controls (HPNE, HPDE), and a PDAC patient-derived organoid at early and late passages; all cell lines were fingerprinted before sequencing.
  • Used 10x Genomics scRNA-seq, scCNV-seq, whole-exome sequencing, snATAC-seq, FISH, ddPCR, inferCNV, clonealign, GSEA, diffusion-map cladogram analysis, and Monocle trajectory inference.
  • Compared cell lines across sources and growth conditions (2D monolayer vs. 3D spheroid), mapped scCNV clones to scRNA clusters, and assessed Moffitt subtype assignment and pathway enrichment.

Limitations

  • The paper states that clonealign’s design filtered out a large number of scCNV-sequenced cells, and that the impact of these findings on rigor and reproducibility is “uncertain, but a matter of concern.”
  • The paper notes that later-passage PDOs may need periodic re-interrogation with single-cell analytics or a reliable surrogate assay to measure divergence over time.
  • Evident from the text: the study profiles a limited panel of cell lines and a single PDO, and several mechanistic interpretations (e.g., AURKA-driven instability in HPDE, BRCA1-mediated genomic quiescence in HPNE) are speculative and not functionally validated here.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/s41467-022-31376-3

Cites

65 of 65 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 64 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    There are marked transcriptomic differences, including expression-based subtype classification, in microsatellite authenticated MiaPaca2 cells obtained from three independent sources.

    “We further demonstrate marked transcriptomic differences (including expression-based subtype classification) in microsatellite authenticated MiaPaca2 cells obtained from three independent sources.”
    Quote in the paperSubstantial claim
  • Findingown data

    A later-passage PDO shows significant genomic and transcriptomic divergence from an earlier passage.

    “Finally, we describe the significant genomic and transcriptomic divergence of a later-passage PDO from an earlier passage”
    Quote in the paperSubstantial claim
  • Findingown data

    Extensive heterogeneity in amplification and deletion profiles on a per-locus basis was uncovered.

    “uncovered extensive heterogeneity in amplification and deletion profiles on a per-locus basis (Supplementary Fig. 4).”
    Quote in the paperSubstantial claim
  • Findingown data

    Transitioning from 2D to 3D spheroid growth models produces transcriptomic shifts.

    “transcriptomic shifts in transitioning from 2D to 3D spheroid growth models”
    Quote in the paperSubstantial claim
  • Findingown data

    Pancreatic cell lines-neoplastic and non-neoplastic-are composed of complex sub-clonal heterogeneity at single cell resolution.

    “We demonstrate that pancreatic cell lines—neoplastic and non-neoplastic—are composed of complex sub-clonal heterogeneity at single cell resolution”
    Quote in the paperSubstantial claim
  • May combine two claims. 45
  • Classified as probably not a substantial claim. 50

  • May combine two claims.

    Findingown data

    All three culture variations of MiaPaCa2 cell lines fall into a common clade, while Panc1 cells branch into their own clade.

    “we observed that all three culture variations of MiaPaCa2 cell lines fall into a common clade (MP2-A, MP2-B, MP2-C), while Panc1 cells, irrespective of growth as monolayer or in 3D, branch into their own clade (Supplementary Fig. 1a, b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    HPNE cells are non-ductal in derivation, with lack of epithelial markers and widespread expression of NES, NOTCH2, and CD44.

    “In contrast to HPDE, HPNE cells are non-ductal in derivation (as demonstrated by lack of epithelial markers, and widespread expression of progenitor cell transcripts NES, NOTCH2 and CD44 on scRNA-seq (Fig. 1b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    High expression of EPCAM and KRT8 was observed in HPAF-II and BxPC3, and VIM was essentially restricted to Panc1 and MiaPaca2.

    “we observed high expression of the epithelial transcripts EPCAM and KRT8 in the previously described “epithelial” lines, HPAF-II and BxPC3, and conversely, the prototypal mesenchymal transcript vimentin (VIM) was essentially restricted to the previously described “mesenchymal” lines Panc1 and MiaPaca2.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    HPNE, Panc1, and MiaPaca2 demonstrated extensive CD44 expression.

    “Notably, HPNE cells, along with Panc1 and MiaPaca2, also demonstrated extensive CD44 expression”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    HPDE cells harbor substantial genomic alterations and a transcriptome that essentially resembles cancer cells.

    “Unexpectedly, HPDE cells harbor substantial genomic alterations and a transcriptome that essentially resembles cancer cells”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    HPNE cells have not evolved a divergent subcellular taxonomy but are homogenous in copy number profiles broadly and at key oncogenic loci.

    “Unlike HPDE cells, scCNV sequencing demonstrates that HPNE cells have not evolved a divergent subcellular taxonomy, but rather are homogenous in their copy number profiles when viewed both broadly and at key oncogenic loci (Fig. 2a, Supplementary Fig. 4a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    HPNE cells retain NES and NOTCH2 markers.

    “HPNE cells, which were originally derived from a nestin (NES) and NOTCH2 expressing pancreatic progenitor cellular population (Lee 2005)13, were confirmed to retain both markers (Fig. 1b).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Lee 2005)13 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    BxPC3 harbors copy number >=4 in nearly all clones at TERT locus, with over 2/3 of cells having TERT copy number more than 1 above mean ploidy.

    “the BxPC3 cell line, which is historically used as a KRAS wild type PDAC cell line, harbors copy number > = 4 in nearly all clones at the TERT gene locus, wherein over 2/3 of cells have a copy number for TERT more than 1 above the mean ploidy (Supplementary Fig. 4b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    HPDE is most transcriptionally similar to HPAF-II and on a distinct clade from HPNE.

    “our scRNAseq interrogation instead shows HPDE is most transcriptionally similar to HPAF-II, and on a distinct clade from the second commonly used “control” line, HPNE (Supplementary Fig. 1b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    HPNE has a benign CNV profile at PDAC loci, with little heterogeneity in copy number across clones.

    “We report that HPNE has a benign CNV profile at these loci, with little heterogeneity in copy number across clones of this culture (Supplementary Fig. 4a).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data

    In Panc1-3D scRNA dataset, subclone 1 was represented by 904 cells and subclone 2 by 223 cells.

    “When we mapped these clonealign-defined unique genomic subclones to specific transcriptional programs across the two datasets, subclone 1 was comprised of 3,251 cells and subclone 2 of 404 cells in Panc1 2D scRNA-seq data (Supplementary Fig. 8c), while in the Panc1-3D scRNA dataset, subclone 1 was represented by 904 cells, and subclone 2 by 223 cells (Supplementary Fig. 8d).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    There was little to no overlap in enriched hallmark pathways (sharing only late estrogen response and cholesterol homeostasis pathways) or oncogenic gene sets (no common pathways) for either of the two subclones between the two growth conditions.

    “There was little to no overlap in the enriched hallmark pathways (sharing only late estrogen response and cholesterol homeostasis pathways) or oncogenic gene sets (no common pathways) for either of the two subclones between the two growth conditions (Supplementary Table 1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    This kind of heterogeneity was not seen within the 2D model, with no significant difference in pathways found between the two subclusters.

    “This kind of heterogeneity was not seen within the 2D model, with no significant difference in pathways found between the two subclusters.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    MP2-B subclone 1 (1096 cells) is characterized by five hallmark pathways, while subclone 2 (813 cells) is defined by six completely distinct gene sets.

    “Subclone 1 of MP2-B (1096 cells) is characterized by five hallmark pathways, while MP2-B subclone 2 (813 cells) is defined by six, completely distinct gene sets (Supplementary Table 1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    MP2-B subclone 1 is enriched for oncogenic pathways LEF1 upregulation, P53, and Cyclin D1, whereas subclone 2 is enriched for MTOR upregulation, EIF4E, RAF, and NEF2L2.

    “We found that subclone 1 is enriched for oncogenic pathways LEF1 upregulation, P53, and Cyclin D1, whereas subclone 2 is enriched for MTOR upregulation, EIF4E, RAF, and NEF2L2 (Supplementary Table 1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    MiaPaca2 lines segregate mostly independently in scRNA-seq, with only a single small cluster where all three overlap.

    “we find that the MiaPaca2 lines segregate mostly independently, with only a single small cluster where all three overlap (Fig. 3a, b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    MP2-B downregulated GSEA hallmark interferon gamma response, and MP2-C has no statistically significant enrichment in hallmark pathways.

    “In contrast, MP2-B downregulated GSEA hallmark interferon gamma response, and MP2-C has no statistically significant enrichment in hallmark pathways based on scRNA-seq GSEA analysis (Fig. 3c).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Upon growth in 3D, Panc1 cells exhibit demonstrable transcriptomic divergence, as well as increased transcriptional diversity, compared to matched 2D culture grown from the same parental vial, upon scRNA-seq analysis.

    “We find here that upon growth in 3D, Panc1 cells exhibit demonstrable transcriptomic divergence, as well as increased transcriptional diversity, compared to matched 2D culture grown from the same parental vial, upon scRNA-seq analysis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    There is phenotypic divergence between assumptively identical MiaPaca2 cultures that appears driven by changes at the RNA level.

    “We report phenotypic divergence between these assumptively “identical” cultures that appears to be driven by changes at the RNA level.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In Panc1 2D scRNA-seq data, subclone 1 comprised 3,251 cells and subclone 2 comprised 404 cells.

    “When we mapped these clonealign-defined unique genomic subclones to specific transcriptional programs across the two datasets, subclone 1 was comprised of 3,251 cells and subclone 2 of 404 cells in Panc1 2D scRNA-seq data (Supplementary Fig. 8c), while in the Panc1-3D scRNA dataset, subclone 1 was represented by 904 cells, and subclone 2 by 223 cells (Supplementary Fig. 8d).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In MP2-C, nearly all cells have copy number >=4 for BLC6, PIK3CA, and BRAF; MP2-B has lower copy number for these genes in a large fraction of cells despite similar overall ploidy.

    “For example, in MP2-C we find that nearly all cells have copy number > = 4 for oncogenes BLC6, PIK3CA, and BRAF. In contrast, MP2-B has lower copy number for these genes in a large fraction of cells, despite similar overall ploidy levels (Supplementary Fig. 4b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    MP2-B has subtype admixture, with 3,456 cells sorting as "basal-like" and 3,556 cells sorting as "classical".

    “We observed that MP2-A and MP2-C have all (9,768 and 10,886 cells respectively) represented by the Moffitt “basal-like” subtype, while MP2-B has subtype admixture, with 3,456 cells sorting as the “basal-like” subtype and 3,556 cells sorting as “classical” (Supplementary Tables 2, 3).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data

    Per sample, the chromosome arms that showed variation in a large percentage of cells were: MP2-B - chr7p, chr10p, chr10q, chr12q; MP2-C - chr5p, chr5q, chr10p, chr10q, chr14q; Panc1-2D - chr14q, chr15q.

    “Per sample, the chromosome arms that showed variation in a large percentage of cells were: MP2-B – chr7p, chr10p, chr10q, chr12q; MP2-C - chr5p, chr5q, chr10p, chr10q, chr14q; Panc1-2D – chr14q, chr15q.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    In Panc1-2D, chr15q mappable regions starting at coordinates <40 Mb or >80 Mb showed a different profile from the other mappable regions.

    “In Panc1-2D, chr15q mappable regions starting at coordinates <40 Mb or >80 Mb, which showed a different profile from the other mappable regions, were also excluded.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Considerable subclonal heterogeneity was identified using single cell analysis in HPDE and HPNE, with an "occult polyclonality" that mirrors PDAC cell lines.

    “We identify considerable subclonal heterogeneity using single cell analysis (with an “occult polyclonality” that mirrors PDAC cell lines), and “major” chromosomal copy number events in HPDE and HPNE that translate to transcriptional upregulation of AURKA and BRCA1, respectively.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    "Major" chromosomal copy number events in HPDE and HPNE translate to transcriptional upregulation of AURKA and BRCA1, respectively.

    “We identify considerable subclonal heterogeneity using single cell analysis (with an “occult polyclonality” that mirrors PDAC cell lines), and “major” chromosomal copy number events in HPDE and HPNE that translate to transcriptional upregulation of AURKA and BRCA1, respectively.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    For MP2-C, there were very low numbers of cells with high confidence clone calls.

    “For MP2-C this was lowered to 50%, due to very low numbers of cells with high confidence clone calls found in this sample.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    In MP2-B, the remaining regions on chr12q outside 93.5 to 126.3 Mb had consistent copy number across cells.

    “In MP2-B, only mappable regions from 93.5 to 126.3 Mb on chr12q were considered, as the remaining regions had consistent copy number across cells.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Culturing a PDAC cell line typically grown in adherent monolayers as a spheroid model alters the underlying chromatin architecture and induces a more diverse transcriptional repertoire.

    “We further show that culturing a PDAC cell line typically grown in adherent monolayers as a spheroid model alters the underlying chromatin architecture and induces a more diverse transcriptional repertoire.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Three independent strains of the same PDAC parental cell line (MiaPaCa2) demonstrably deviate from one another on single cell analysis, both in terms of underlying transcriptional profiles and the outgrowth of unique genomic subclones.

    “In line with this observation, we report that three independent strains of the same PDAC parental cell line (MiaPaCa2) demonstrably deviate from one another on single cell analysis, both in terms of underlying transcriptional profiles and the outgrowth of unique genomic subclones.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The earlier passage PDO has demonstrable expression of "basal-like" transcripts but these did not overlap with the "basal-like" transcripts expressed by the later passage PDO.

    “The earlier passage PDO has demonstrable expression of “basal-like” transcripts (Fig. 6e) but these did not overlap with the “basal-like” transcripts expressed by the later passage PDO (Fig. 6e).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    For MP2-C, cells called as clone 1 or clone 3 showed very similar expression.

    “This was because cells called as either of these clones showed very similar expression.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    BxPC3 TERT copy number was confirmed via FISH.

    “BxPC3 TERT copy number was also confirmed via fluorescence in situ hybridization (FISH)”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Early passages of PDAC PDOs maintain genomic and transcriptomic features of the tumor of origin and harbor a gene signature of response to commonly used cytotoxic agents, enabling therapeutic prediction.

    “Encouragingly, early passages of PDAC PDOs maintain genomic and transcriptomic features of the tumor of origin, and harbor a gene signature of response to commonly used cytotoxic agents, enabling therapeutic prediction7,8.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [7] Aung, K. L. et al. Genomics-driven precision medicine for advanced pancreatic cancer: early results from the COMPASS trial. Clin. Cancer Res 24, 1344–1354 (2018). source

      Not checked in the free run.

    • cites [8] Raghavan, S. et al. Microenvironment drives cell state, plasticity, and drug response in pancreatic cancer. Cell 184, 6119–6137 e6126 (2021). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Aurora kinase A is commonly overexpressed in pancreatic cancers and contributes to chromosomal instability and tumor progression.

    “The encoded protein Aurora kinase A is commonly overexpressed in pancreatic cancers, and contributes to both chromosomal instability through destabilization of the mitotic spindle assembly and towards tumor progression via phosphorylation of substrate proteins19,20.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [19] Furukawa, T. Impacts of activation of the mitogen-activated protein kinase pathway in pancreatic cancer. Front. Oncol. 5 (2015). source

      Not checked in the free run.

    • cites [20] Li, D. et al. Overexpression of oncogenic STK15/BTAK/Aurora A kinase in human pancreatic cancer. Clin. Cancer Res 9, 991–997 (2003).

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    KRAS wild type PDAC, including BxPC3, acquire in-frame deletions of BRAF that result in constitutive activation of the kinase domain.

    “It is known that in contrast to melanoma, cases of KRAS wild type PDAC, including the BxPC3 cell line itself, acquire in-frame deletions of BRAF that result in constitutive activation of the kinase domain (Chen 2016)15.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Chen 2016)15 source

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Ben-David et al. identified widespread genomic and transcriptomic variabilities in single cell clones isolated and expanded from the same parental cancer cells, which translated into differences in drug responsiveness in vitro.

    “A recent seminal publication by Ben-David et al.6 identified widespread genomic and transcriptomic variabilities in single cell clones isolated and expanded from the same parental cancer cells, which further translated into differences in drug responsiveness in vitro”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Ben-David et al.6 Ben-David, U. et al. Genetic and transcriptional evolution alters cancer cell line drug response. Nature 560, 325–330 (2018). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    HPNE lacks a ductal transcriptional profile and is enriched in markers associated with progenitor populations of uncertain histogenesis, which need to be factored in the use of these cells as an appropriate control for PDAC cells.

    “Ultimately, while HPNE does not exhibit the subclonal heterogeneity observed with HPDE, its lack of a ductal transcriptional profile and enrichment in markers associated with progenitor populations of uncertain histogenesis need to be factored in the use of these cells as an appropriate control for PDAC cells.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Previous work challenged the assumption that cell lines are clonal, stable entities and explored phenotypic variability between HeLa and MCF7 cell lines via cross-laboratory multi-omic comparisons.

    “Previous work challenged the long-maintained assumption in cancer biology research that cell lines are clonal, stable entities, and explored the existence of phenotypic variability between widely used HeLa5 and MCF76 cell lines via cross-laboratory multi-omic comparisons.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites HeLa5 and MCF76

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All samples were submitted for fingerprinting prior to sequencing to confirm cell line identity.

    “All samples were submitted for fingerprinting via MD Anderson Core Facilities prior to sequencing to confirm cell line identity.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Brca1 is a component of homologous recombination repair machinery, and BRCA1 is uncommonly mutated in PDAC (~1%), typically germline, and associated with HRR defective cancers.

    “The encoded Brca1 protein is a component of the homologous recombination repair (HRR) machinery, and BRCA1 is uncommonly mutated in PDAC (~1%), typically in the germline, and associated with HRR defective cancers22,23.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [22] Yoshida, K. & Miki, Y. Role of BRCA1 and BRCA2 as regulators of DNA repair, transcription, and cell cycle in response to DNA damage. Cancer Sci. 95, 866–871 (2004). source

      Not checked in the free run.

    • cites [23] Nguyen, L. et al. Pan-cancer landscape of homologous recombination deficiency. Nat. Commun. 11, 5584 (2020). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    2D monolayers might lack critical features associated with in vivo tumor progression, such as hypoxia.

    “Additional work has also shown that 2D monolayers might lack critical features associated with in vivo tumor progression, such as hypoxia41,42.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [41] Loessner, D. et al. Bioengineered 3D platform to explore cell-ECM interactions and drug resistance of epithelial ovarian cancer cells. Biomaterials 31, 8494–8506 (2010). source

      Not checked in the free run.

    • cites [42] Riedl, A. et al. Comparison of cancer cells in 2D vs 3D culture reveals differences in AKT-mTOR-S6K signaling and drug responses. J. Cell Sci. 130, 203–218 (2017).

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Knockdown of LGALS1 in vitro reduced cell migration and invasion capabilities in pancreatic cancer associated cells.

    “knockdown of LGALS1 in vitro reduced cell migration and invasion capabilities in pancreatic cancer associated cells34.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [34] Chen, R. et al. Proteins associated with pancreatic cancer survival in patients with resectable pancreatic ductal adenocarcinoma. Lab Invest 95, 43–55 (2015). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Both MP2-B subclones were found at relatively high frequency, suggesting neither has a clear fitness advantage over the other.

    “As both subclones were found at relatively high frequency (Fig. 4e), it would suggest that neither has a clear fitness advantage over the other.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Subsequent analysis revealed extensive inter-laboratory heterogeneity with respect to phenotype, copy number variations, and drug response experiments.

    “Subsequent analysis revealed extensive inter-laboratory heterogeneity with respect to phenotype, copy number variations, and even drug response experiments”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S2 (checkable). May combine two claims.

    Interpretationreasoning

    This finding may impact how prevalent PDAC classification schemes are applied to monolayer culture models and any subtype-based therapeutic outcome predictions derived from these that are then extrapolated to patients.

    “This finding may impact how prevalent PDAC classification schemes are applied to monolayer culture models and any subtype-based therapeutic outcome predictions derived from these that are then extrapolated to patients.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Primary tumors have been shown to undergo subtype switching in response to microenvironmental cues.

    “primary tumors have been shown to undergo subtype switching in response to microenvironmental cues (Torres 2018)37.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Torres 2018 source

      Not checked in the free run.

    • cites [37] Torres, C. & Grippo, P. J. Pancreatic cancer subtypes: a roadmap for precision medicine. Ann. Med. 50, 277–287 (2018). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    LGALS1 overexpression has been negatively associated with survival in PDAC patients.

    “LGALS1 overexpression has also been negatively associated with survival in PDAC patients33”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [33] Chen, R. et al. Stromal galectin-1 expression is associated with long-term survival in resectable pancreatic ductal adenocarcinoma. Cancer Biol. Ther. 13, 899–907 (2012). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    KRT13 expression has previously been shown to predict worse overall survival in pancreatic cancer.

    “This gene has previously been shown to predict worse overall survival in pancreatic cancer32.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [32] Birnbaum, D. J. et al. A 25-gene classifier predicts overall survival in resectable pancreatic cancer. BMC Med. 15, 170 (2017). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    PDAC transcriptional subtypes are known to associate with specific microenvironmental niches.

    “PDAC transcriptional subtypes are known to associate with specific microenvironmental niches (Raghavan 2021)8”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Raghavan 2021 source

      Not checked in the free run.

    • cites [8] Raghavan, S. et al. Microenvironment drives cell state, plasticity, and drug response in pancreatic cancer. Cell 184, 6119–6137 e6126 (2021). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Campbell et al. developed the clonealign algorithm, which established a framework for mapping genomic-transcriptome associations in single-cell datasets derived from the same parental culture but generated using individual assays.

    “Building on this finding, Campbell et al. developed the clonealign algorithm31, which established a framework for how genomic-transcriptome associations may be mapped in single-cell datasets derived from the same parental culture, but generated using individual assays.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Campbell et al.31 Campbell, K. R. et al. clonealign: statistical integration of independent single-cell RNA and DNA sequencing data from human cancers. Genome Biol. 20, 54 (2019). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Ex vivo 3D models of tumor growth are being increasingly adopted, as a number of comparative studies between 3D spheroids and 2D monolayer cultures have shown that 3D culture more faithfully recapitulates in vivo biology.

    “Ex vivo 3D models of tumor growth are being increasingly adopted, as a number of comparative studies between 3D spheroids and 2D monolayer cultures have shown that 3D culture more faithfully recapitulates in vivo biology38–40.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [38] Zeeberg, K. et al. Assessment of different 3D culture systems to study tumor phenotype and chemosensitivity in pancreatic ductal adenocarcinoma. Int J. Oncol. 49, 243–252 (2016). source

      Not checked in the free run.

    • cites [39] Lazzari, G. et al. Multicellular spheroid based on a triple co-culture: a novel 3D model to mimic pancreatic tumor complexity. Acta Biomater. 78, 296–307 (2018). source

      Not checked in the free run.

    • cites [40] Longati, P. et al. 3D pancreatic carcinoma spheroids induce a matrix-rich, chemoresistant phenotype offering a better model for drug testing. BMC Cancer 13, 95 (2013). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    NEAT1 has been shown to promote PDAC growth and metastasis and predict poor prognosis.

    “This gene has also been shown to promote PDAC growth and metastasis and predict poor prognosis (Feng 2020)35.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [35] Feng, Y. et al. LncRNA NEAT1 facilitates pancreatic cancer growth and metastasis through stabilizing ELF3 mRNA. Am. J. Cancer Res. 10, 237–248 (2020).

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The dichotomous Moffitt classification schema of "basal-like" and "classical" subtypes was initially established using bulk RNA sequencing strategies applied to human tumor samples.

    “The dichotomous Moffitt classification schema of “basal-like” and “classical” subtypes was initially established using bulk RNA sequencing strategies applied to human tumor samples (Moffitt 2015)36.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Moffitt 2015 source

      Not checked in the free run.

    • cites [36] Moffitt, R. A. et al. Virtual microdissection identifies distinct tumor- and stroma-specific subtypes of pancreatic ductal adenocarcinoma. Nat. Genet. 47, 1168–1178 (2015). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Many genes display copy-number gene-dosage effects on transcriptional expression.

    “Previous work in which DNA and RNA were assayed in the same cells has shown that many genes display copy-number gene-dosage effects on transcriptional expression28–30.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [28] Macaulay, I. C. et al. G&t-seq: parallel sequencing of single-cell genomes and transcriptomes. Nat. Methods 12, 519 (2015). source

      Not checked in the free run.

    • cites [29] Dey, S. S. et al. Integrated genome and transcriptome sequencing of the same cell. Nat. Biotechnol. 33, 285 (2015). source

      Not checked in the free run.

    • cites [30] Han, K. Y. et al. SIDR: simultaneous isolation and parallel sequencing of genomic DNA and total RNA from single cells. Genome Res. 28, 75–87 (2018). source

      Not checked in the free run.

  • May combine two claims.

    Study design and conductown data

    InferCNV confirmed clonealign results, with the majority of cells in each condition showing lower copy number in chr15 compared to chr14 and a small subset of cells showing the reverse.

    “This confirmed the clonealign results, with the majority of (but not all) cells in each condition showing lower copy number in chr15 compared to chr14 and a small subset of cells showing the reverse.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Tickle 2019

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The MiaPaca2 cell line was initially generated from a primary PDAC lesion of a 65-year-old male with tumor involving pancreatic body, tail, and periaortic invasion.

    “The MiaPaca2 cell line was initially generated from a primary PDAC lesion of a 65-year-old male, where the tumor had involved the pancreatic body, tail, and demonstrated periaortic invasion24.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [24] Deer, E. L. et al. Phenotype and genotype of pancreatic cancer cell lines. Pancreas 39, 425–435 (2010). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    A recent assessment for "basal-like" and "classical" cells in PDAC tissues by subtype-specific protein markers has uncovered bi-phenotypic "hybrid" cells.

    “Of note, a recent assessment for “basal-like” and “classical” cells in PDAC tissues by subtype-specific protein markers has uncovered bi-phenotypic “hybrid” cells51, while Ting and colleagues have shown evidence of subtype “drift” in PDAC tissues with various therapies52, both lines of evidence supporting the plasticity of subtype states.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [51] Williams, H. L. et al. Abstract PR-006: Spatially resolved, single cell assessment of pancreatic ductal adenocarcinoma expression subtypes reveals mixed and hybrid basal-classical marker expression with prognostic significance and discrete spatial localization. Cancer Res. 80: PR-006-PR-006 (2020). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Cells with ploidy more than one standard deviation from the mean across cells were removed.

    “Cells with ploidy more than one standard deviation from the mean across cells were removed.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    In rescaled diffusion space, phenotype distance between cells is more faithfully represented.

    “we first map the cells from high dimensional transcriptomic space to a low dimensional rescaled diffusion space (Setty 2019)61, where phenotype distance between cells are more faithfully represented”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Setty 2019 source

      Not checked in the free run.

    • cites [61] Setty, M. et al. Characterization of cell fate probabilities in single-cell data with Palantir. Nat. Biotechnol. 37, 451–460 (2019). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    A patient was recruited through informed written consent following institutional review board (IRB) approval.

    “A patient was recruited at the University of Texas MD Anderson Cancer Center through informed written consent following institutional review board (IRB) approval (Human Research Protection Program Lab00-396 and PA15-0014).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    For scRNA analysis of cell lines, cells with less than 20% mitochondrial gene expression were retained.

    “For removal of mitochondrial genes in cell lines, we subsetted all cells with less than 20% mitochondrial gene expression.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Ting and colleagues have shown evidence of subtype "drift" in PDAC tissues with various therapies.

    “Of note, a recent assessment for “basal-like” and “classical” cells in PDAC tissues by subtype-specific protein markers has uncovered bi-phenotypic “hybrid” cells51, while Ting and colleagues have shown evidence of subtype “drift” in PDAC tissues with various therapies52, both lines of evidence supporting the plasticity of subtype states.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [52] Porter, R. L. et al. “Epithelial to mesenchymal plasticity and differential response to therapies in pancreatic ductal adenocarcinoma. Proc. Natl Acad. Sci. USA 116, 26835–26845 (2019). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Rescaling the diffusion coordinates by their eigenvalues can circumvent the need to choose the optimal number of diffusion coordinates.

    “However, it has been proposed that rescaling the diffusion coordinates by their eigenvalues can circumvent this issue61.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [61] Setty, M. et al. Characterization of cell fate probabilities in single-cell data with Palantir. Nat. Biotechnol. 37, 451–460 (2019). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Cells listed as noisy or with a haploid genome (mean ploidy < 1.5) were excluded from scCNV clonal analysis.

    “Cells listed as noisy or with a haploid genome (mean ploidy < 1.5) were excluded”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Quality control on nuclei for snATAC analysis was done by quantifying nucleosome signal, TSS enrichment, number of peaks in fragmentation regions, and nucleosome signal according to Signac software recommendations (TSS Enrichment > 2, nucleosome signal < f10, blacklist ratio < 0.05, percent reads in peaks > 15, peak region fragments between 1,000 and 20,000).

    “Quality control on nuclei was done by quantifying nucleosome signal, TSS enrichment, number of peaks in fragmentation regions, and nucleosome signal according to Signac software recommendations (TSS Enrichment > 2, nucleosome signal < f10, blacklist ratio < 0.05, percent reads in peaks > 15, peak region fragments between 1,000 and 20,000).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Cells with higher expression of mitochondrially encoded genes were filtered out post hoc based on the results of the clustering.

    “Cells with higher expression of mitochondrially encoded genes were filtered out post hoc based on the results of the clustering.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Interpretationown data

    The level of subclonal heterogeneity observed on single cell analysis here is unexpected.

    “While established PDAC models are assumptively oligoclonal and reflect human tumors on a global level based on published data, the level of subclonal heterogeneity observed on single cell analysis here is unexpected, leading us to propose the phrase “occult polyclonality” as a molecular paradigm upon which research involving cell lines could be predicated.”
    Quote in the paperSubstantial claim
    • The authors state this as an assumption: Established PDAC models are assumptively oligoclonal and reflect human tumors on a global level based on published data.

  • May combine two claims.

    Interpretationreasoning

    It calls into question the assumptions behind isogenic perturbation experiments in cultured cell lines, or the use of archived, cell line-specific profiling data for drawing inferences that are temporally disparate from the data generation point.

    “For example, it calls into question the assumptions behind isogenic perturbation experiments in cultured cell lines, or the use of archived, cell line-specific profiling data for drawing inferences that are temporally disparate from the data generation point.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Diffusion maps transform data from high dimensional gene expression space to a space of much lower dimensions, where usual Euclidean distance is reflective of the cellular phenotypic distance.

    “Essentially, they transform data from high dimensional gene expression space to a space of much lower dimensions, where usual Euclidean distance is reflective of the cellular phenotypic distance.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [62] Lafon, S. et al. Data fusion and multicue data matching by diffusion maps. IEEE Trans. Pattern Anal. Mach. Intell. 28, 1784–1797 (2006). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    All libraries were assessed for quality on Agilent TapeStation 2200 and quantified using both Agilent TapeStation 2200 reagents and Qubit dsDNA HS Reagent kits before loading onto NextSeq500.

    “All libraries were assessed for quality on Agilent TapeStation 2200 and quantified using both Agilent TapeStation 2200 reagents and Qubit dsDNA HS Reagent kits (Invitrogen Cat. Q33231) before loading onto NextSeq500 (Illumina).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    PD01late shows true subtype admixture (some cells classify as basal and others as classical) not due solely to admixture within any given cell, suggesting gradual transcriptional reprogramming in the PDO over time.

    “We confirmed that this mix of classical and basal genes in PD01late is not due solely to admixture within any given cell, and show that some cells classify as basal and others as classical, resulting in a true subtype admixture that suggests gradual transcriptional reprogramming in the PDO over time (Supplementary Fig. 10).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Our data are comparable to the recent findings of Navin, Michor and colleagues, who identified between seven to 22 subclones on scCNV analysis of triple negative breast cancer tumors and cell lines.

    “Our data are comparable to the recent findings of Navin, Michor and colleagues, who identified between seven to 22 subclones on scCNV analysis of triple negative breast cancer tumors and cell lines (Minussi 2021)53.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Minussi 2021

      Not checked in the free run.

    • cites [53] Minussi, D. C. et al. Breast tumours maintain a reservoir of subclonal diversity during expansion. Nature (2021). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Mycoplasma testing was performed on media from each cell line.

    “A volume of 3 mL of media from each cell line was harvested and sent to the MD Anderson core facilities for mycoplasma testing.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Cells defined as noisy in the QC summary file were removed prior to downstream scCNV analysis.

    “Cells defined as noisy in the QC summary file (“per_cell_summary_metrics.csv”) were removed prior to downstream analysis as well.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Cells containing less than 200 genes were removed from PDO single-cell analysis.

    “Cells containing less than 200 genes were removed”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Cancer cells retain a "reservoir" of subclonal diversity, such that isolated single cell clones re-diversify their genomes within a relatively short time frame, at a calculated pace of one CNV event every four cell divisions.

    “Notably, these investigators also reported that cancer cells retain a “reservoir” of subclonal diversity, such that isolated single cell clones re-diversify their genomes within a relatively short time frame, at a calculated pace of one CNV event every four cell divisions.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [53] Minussi, D. C. et al. Breast tumours maintain a reservoir of subclonal diversity during expansion. Nature (2021). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Molecular fingerprinting was performed on a portion of cells.

    “A portion of cells were sent to the MD Anderson core facility for molecular fingerprinting, and all cell lines were counted using a Countess II automated cell counter.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

    Interpretationreasoning

    The report by Navin, Michor and colleagues in breast cancer, and our own data in PDAC, harbors the potential to have a profound impact on the rigor and reproducibility of research, both within and across laboratories.

    “The report by Navin, Michor and colleagues in breast cancer, and our own data in PDAC, harbors the potential to have a profound impact on the rigor and reproducibility of research, both within and across laboratories.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    PDOs have emerged as a promising preclinical model for therapeutic prediction and functional studies across multiple cancer types, including in PDAC.

    “PDOs have emerged as a promising preclinical model for therapeutic prediction and functional studies across multiple cancer types, including in PDAC18,19,48–50.”
    Quote in the paperNot a substantial claim0 of 5 sources checked
    • cites [18] Radulovich, N. et al. Human pancreatic duct epithelial cell model for KRAS Transformation. Methods Enzymol., Academic Press. 439, 1–13 (2008). source

      Not checked in the free run.

    • cites [19] Furukawa, T. Impacts of activation of the mitogen-activated protein kinase pathway in pancreatic cancer. Front. Oncol. 5 (2015). source

      Not checked in the free run.

    • cites [48] Jacob, F. et al. A patient-derived glioblastoma organoid model and biobank recapitulates inter- and intra-tumoral heterogeneity. Cell 180, 188–204 e122 (2020). source

      Not checked in the free run.

    • cites [49] Ganesh, K. et al. A rectal cancer organoid platform to study individual responses to chemoradiation. Nat. Med. 25, 1607–1614 (2019). source

      Not checked in the free run.

    • cites [50] Ooft, S. N. et al. Patient-derived organoids can predict response to chemotherapy in metastatic colorectal cancer patients. Sci. Transl. Med. 11 (2019). source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The study was conducted in accordance with Good Clinical Practices concerning medical research in humans per the Declaration of Helsinki.

    “The study was conducted in accordance with Good Clinical Practices concerning medical research in humans per the Declaration of Helsinki.”
    Quote in the paperNot a substantial claim
81 of 81

  • The paper relies on this without support.

    It is not well established whether cell lines can be reliably classified into Moffitt subtypes as freshly dissociated tumor tissue or PDO models, nor whether subtype admixture would be observable when single-cell methods are applied to subclonal derivatives of parental cell lines.

    “However, it is not well established whether cell lines can reliably classified into Moffitt subtypes as freshly dissociated tumor tissue or PDO models, nor whether there would be an observable degree of subtype admixture when single-cell methods are applied to subclonal derivatives of parental cell lines.”

  • The paper relies on this without support.

    Within single-cell analytics, there is scant data on the extent of transcriptional adaptations that occur when modifying conventional monolayer culture to a 3D spheroid model.

    “Within the context of single-cell analytics, there is scant data to address the extent of transcriptional adaptations that occur in the process of modifying “conventional” monolayer culture to a 3D spheroid model.”

  • The paper relies on this without support.

    Whether the transcriptome of PDOs evolves over time in culture is less well studied.

    “However, whether the transcriptome of PDOs evolves over time in culture is less well studied.”

  • The authors state this as an assumption.

    Established PDAC models are assumptively oligoclonal and reflect human tumors on a global level based on published data.

    “While established PDAC models are assumptively oligoclonal and reflect human tumors on a global level based on published data, the level of subclonal heterogeneity observed on single cell analysis here is unexpected, leading us to propose the phrase “occult polyclonality” as a molecular paradigm upon which research involving cell lines could be predicated.”

  • The paper relies on this without support.

    Early passages of PDOs retain the clonal genomic alterations observed in the parental tumor, as well as the transcriptomic signature(s) predictive of response to cytotoxic agents.

    “Early passages of PDOs retain the clonal genomic alterations observed in the parental tumor, as well as the transcriptomic signature(s) predictive of response to cytotoxic agents.”

  • The paper relies on this without support.

    Recently developed transcriptome-based subtyping of PDAC have demonstrated both predictive and prognostic significance in early studies.

    “Recently developed transcriptome-based subtyping of PDAC have demonstrated both predictive and prognostic significance in early studies.”

  • The paper relies on this without support.

    Certain dependencies (such as metabolic dependencies) initially identified in monolayer conditions have not subsequently been validated within an in vivo context.

    “While we do not posit that one culture condition is necessarily more reflective of in vivo biology than the other, it is noteworthy that certain dependencies (such as metabolic dependencies) initially identified in monolayer conditions have not subsequently been validated within an in vivo context.”

  • The paper relies on this without support.

    A single cell can be labeled as belonging to the subtype with the highest average expression.

    “We then reasoned that a single cell can be labeled as belonging to the subtype with the highest average expression.”
8 of 8

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Single-cell RNA sequencing of human and murine tumors found several distinct neoplastic epithelial populations in one tumor, corresponding to multiple PDAC subtypes.

    “scRNA-seq datasets from human and murine tumors have identified multiple unique neoplastic epithelial populations within individual tumors149,269, which represent multiple PDAC subtypes within the same tumor270.”

    From Pancreatic cancer: Advances and challenges (2023) · cites this paper as [270]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • KRT8 is assigned to both epithelial and mesenchymal markers

    Minor

    The Results identify KRT8 as an epithelial transcript expressed in epithelial cell lines, but the Fig. 1 caption labels it a marker of mesenchymal cellular origin.

1 of 1

Cites

Flagged first, then by citations

Research works printed but not linked: 3

  1. McInnes, L. & Healy, J. UMAP: uniform manifold approximation and projection for dimension reduction. ArXiv e-prints 1802, 03426 (2018).
  2. Riedl, A. et al. Comparison of cancer cells in 2D vs 3D culture reveals differences in AKT-mTOR-S6K signaling and drug responses. J. Cell Sci. 130, 203–218 (2017).
  3. Korotkevich, G. et al. Fast gene set enrichment analysis (2021). bioRxiv: 060012.

Cited by

Sorted by citations

Related

Authors
Monberg, M. E., Geiger, H., Lee, J. J., Sharma, R., Semaan, A., Bernard, V., Wong, J., Wang, F., Liang, S., Swartzlander, D. B., Stephens, B. M., Katz, M. H. G., Chen, K., Robine, N., Guerrero, P. A., & Maitra, A.
Affiliations
  • The University of Texas MD Anderson Cancer Center (US)
  • New York Genome Center (US)
Corresponding author
Monberg, Maria E.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2022-06-25
Volume
13
Issue
1
Pages
3652-3652
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-022-31376-3
PMC
PMC9233687
OpenAlex
W4283451026
Semantic Scholar
7462779075be288c8e8060dedffb9912d4604284
Cited by
30
References
65
Citations by year
2026: 6 · 2025: 3 · 2024: 6 · 2023: 9 · 2022: 4 · 2021: 2
FWCI
2.32
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Digital Commons-TMC (Texas Medical Center) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • Foundation for the National Institutes of Health (T32CA009599, U54CA096300, U54CA096297, T32CA217789, NIH U01CA196403, NIH U01CA200468, P50CA221707)
  • Deutsche Forschungsgemeinschaft (SE-2616/2-1)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer Genomics and Diagnostics
  • Health Sciences › Medicine › Oncology › Pancreatic and Hepatic Oncology Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
Keywords
pancreatic ductal adenocarcinoma, polyclonality, tumor heterogeneity, patient-derived organoids, copy number variation, 3D spheroid culture, in vitro evolution, reproducibility, preclinical cancer models
MeSH terms
Humans, Pancreas, Pancreatic Neoplasms, Reproducibility of Results, Carcinoma, Pancreatic Ductal, Cell Line, Tumor, DNA Copy Number Variations

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-06.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-06.

Statistics and arithmetic are recomputed, not judged.

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