Organoid cultures derived from patients with advanced prostate cancer

Authors

Dong Gao

Ian Vela

Andrea Sboner

Phil J. Iaquinta

Wouter R. Karthaus

Anuradha Gopalan

Himisha Beltran

Juan-Miguel Mosquera

Mark A. Rubin

Howard I. Scher

Hans Clevers

Charles L. Sawyers

Yu Chen

Doi

10.1016/j.cell.2014.08.016

PMID: 25201530 · DOI: 10.1016/j.cell.2014.08.016 · Journal: Cell (2014)

TL;DR

Gao et al. established seven patient-derived 3D organoid lines (MSK-PCa1–7) from metastatic biopsies (n=6 from 32 metastasis specimens; ~15–20% efficiency) and one circulating-tumor-cell sample from men with advanced prostate cancer at Memorial Sloan Kettering. Whole-exome sequencing, array-CGH, and RNA-seq showed the lines recapitulate the molecular and phenotypic diversity of CRPC — including TMPRSS2-ERG fusion, SPOP mutation, SPINK1 overexpression, CHD1 loss, FOXA1 and PIK3R1 mutations, biallelic PTEN loss, and frequent loss of TP53/RB pathway function — and they remain amenable to in vitro and xenograft drug testing (enzalutamide, everolimus, BKM-120). The work effectively doubled the number of prostate cancer cell lines available and produced the first in vitro model carrying a SPOP F133L hotspot mutation.

Cohort & data

  • Seven patient-derived organoid lines from men with advanced PRAD; six from metastatic biopsies (bone, lymph node, soft tissue, pleural effusion), one (MSK-PCa5) from circulating tumor cells of a CRPC patient with >100 CTCs / 8 mL blood.
  • Source biopsy material: 32 metastasis samples (18 bone, 9 lymph node, 2 liver, 1 brain, 1 bladder, 1 pleural effusion) and 17 blood draws collected Dec 2012 – Nov 2013 at Memorial Sloan-Kettering Cancer Center under IRB 90-040 / 06-107.
  • Assays per line: whole-exome sequencing (whole-exome-seq) on Agilent SureSelectXT2 (~250× nominal, mean ~142×), RNA-seq (rna-seq) on Illumina HiSeq, copy-number profiling by Agilent SurePrint G3 1M array-CGH (array-cgh-agilent-1m), histology and immunohistochemistry (immunohistochemistry), in vitro drug screens (organoid-drug-screening), and CB17 SCID mouse xenografts (subcutaneous-xenograft).
  • Comparison cohorts queried via the cBioPortal: Taylor 2010, Grasso 2012, Barbieri 2012, and Baca 2013 prostate datasets. Organoid line data deposited as prad_mskcc_cheny1_organoids_2014.

Key findings

  • Establishment efficiency for continuously propagated organoid lines was ~15–20% from metastatic biopsies (6/32 attempts); short-term cultures held for 1–2 months in ~70% of soft-tissue and ~30% of bone biopsies before being overtaken by stromal or normal epithelial cells PMID:25201530.
  • Somatic non-synonymous SNV+indel counts ranged from 29 (MSK-PCa1) to 75 (MSK-PCa4) with a mean of 45.4 per line — consistent with reported CRPC mutational burden.
  • The lines captured stereotypic prostate cancer copy-number changes (gains of chr 7 and 8q; losses of 5q, 6q, 8p, 13q, 18q). MSK-PCa1 and MSK-PCa3 carry the TMPRSS2-ERG interstitial deletion; MSK-PCa1 and MSK-PCa7 carry focal heterozygous deletion at 3p21 spanning candidate tumor suppressors FOXP1, SHQ1, and RYBP.
  • Three lines (MSK-PCa2, MSK-PCa4, MSK-PCa7) carry focal homozygous deletion of CHD1 with complete loss of protein expression.
  • 6/7 lines carry focal homozygous deletion of PTEN or its promoter; MSK-PCa2 also harbors AR amplification (a feature found in ~50% of CRPC).
  • TP53 is mutated in 4/7 organoids with complete loss of p53 function (either single-copy loss or copy-neutral LOH); FOXA1 and PIK3R1 mutations are also present and expressed at high RPKM.
  • MSK-PCa7 carries a heterozygous SPOP F133L substrate-recognition-domain hotspot mutation — the first in vitro model of this primary-prostate-cancer mutation.
  • Additional mutations seen across the panel: ATRX, CHEK2 (genomic stability), MED1, KDM4C, KDM4D, KMT2D (reported as MLL2), SETDB1, SETD2 (chromatin modifiers), and tumor suppressors TSC2 and CDK12.
  • Concordance with original tumor tissue was high: in matched WES of MSK-PCa2 and MSK-PCa7 only 4 and 7 discrepant calls respectively (all attributable to low coverage or repeats); RNA-seq allele frequencies and gene-expression profiles of organoid vs tumor were tightly correlated in MSK-PCa6.
  • Phenotypic spectrum across the seven lines: AR-dependent adenocarcinoma (MSK-PCa2, MSK-PCa7), AR-negative adenocarcinoma, AR-low squamous-differentiated tumor (MSK-PCa6), and treatment-induced neuroendocrine prostate cancer (MSK-PCa4, SYP+/CHGA+/CD56+, no AR, no cytokeratin). MSK-PCa5 and MSK-PCa7 express SPINK1 (mutually exclusive with ETS fusions).
  • Biallelic PTEN loss occurred in all six CRPC-derived lines but not in the hormone-sensitive MSK-PCa7; in a re-analysis of Grasso 2012 CRPC samples, PTEN function was lost in 30/36 CRPC cases vs ~10% of primary tumors, and TP53 was inactivated in 27/36 CRPC cases.
  • RB1: heterozygous loss with no detectable protein in MSK-PCa1, MSK-PCa4, and MSK-PCa5; RNA-seq detected a complete RB1 deletion in MSK-PCa5 missed by array-CGH and WES. Re-examination of Grasso 2012 CRPC suggested homozygous RB loss / reduced expression in ~70% of cases. Two lines without RB1 alterations (MSK-PCa3, MSK-PCa6) instead carry biallelic loss of CDKN2A, producing hyperphosphorylated RB via unrestrained CDK4/6.
  • Drug response: AR-amplified MSK-PCa2 was exquisitely sensitive to enzalutamide (IC50 ~50 nM) in vitro and in CB17 SCID xenografts; other lines were resistant. MSK-PCa2 (PTEN loss + PIK3R1 mutation) was also sensitive to everolimus and BKM-120 (buparlisib). In MSK-PCa1 and MSK-PCa2 xenografts everolimus slowed growth without shrinkage; in MSK-PCa2 everolimus + enzalutamide combined significantly enhanced response over enzalutamide alone.

Genes & alterations

  • AR — amplification in MSK-PCa2 (mirrors ~50% of CRPC); silencing in MSK-PCa1; high-level expression with AR target-gene programs in MSK-PCa2/PCa7.
  • TMPRSS2–ERG — interstitial deletion fusion in MSK-PCa1 (non-expressed, AR-negative line) and MSK-PCa3 (expressed).
  • SPOP — heterozygous F133L hotspot in MSK-PCa7; first in vitro model.
  • CHD1 — focal homozygous deletion with complete protein loss in MSK-PCa2, MSK-PCa4, MSK-PCa7.
  • PTEN — homozygous deletion of gene or promoter in 6/7 lines; biallelic loss in all CRPC lines.
  • TP53 — mutation with complete loss of WT allele in 4/7 lines.
  • FOXA1 and PIK3R1 — expressed mutations seen in CRPC; MSK-PCa2 PIK3R1 mutation co-occurs with PTEN loss and PI3K-inhibitor sensitivity.
  • RB1 — heterozygous loss with no detectable protein in 3 lines; complete deletion in MSK-PCa5 found only by RNA-seq.
  • CDKN2A — biallelic loss in MSK-PCa3 and MSK-PCa6 as alternative RB-pathway hit.
  • SHQ1, RYBP, FOXP1 — 3p21 focal heterozygous deletion in MSK-PCa1 and MSK-PCa7.
  • SPINK1 — overexpression in MSK-PCa5 and MSK-PCa7 (ETS-fusion exclusive subtype).
  • ATRX, CHEK2 — genomic-stability mutations.
  • MED1, KDM4C, KDM4D, KMT2D, SETDB1, SETD2 — chromatin-modifier mutations consistent with Grasso 2012 CRPC findings.
  • TSC2, CDK12 — tumor-suppressor mutations.

Clinical implications

  • AR amplification predicts enzalutamide sensitivity in this panel; the authors propose combination AR + PI3K-pathway blockade (enzalutamide + everolimus) for tumors with concurrent AR activation and PTEN/PIK3R1 alterations, based on MSK-PCa2 xenograft data.
  • Near-universal loss of PTEN, TP53, and RB pathway function in CRPC-derived organoids supports prioritizing therapies that target these pathways (e.g., CDK4/6 inhibition in CDKN2A-deleted, RB-intact tumors).
  • Patient-derived organoid platform is presented as a route to closing the prostate-cancer cell-line gap and as a substrate for prospective drug testing during precision-medicine trials that biopsy metastatic lesions.

Limitations & open questions

  • Authors note only ~15–20% derivation efficiency; many cultures were overtaken by normal liver or lung epithelial cells (some “organoid lines” lacked CNAs and were re-classified as normal-origin), highlighting need for tumor-cell selection strategies.
  • Some mutations may have been acquired during culture; in archival lymph-node FFPE comparisons only 65–67% of organoid point mutations were detectable in the matched prior metastasis, leaving open whether new mutations arose in vivo (during hormone-sensitive → CRPC transition) or in vitro.
  • Small sample size (n=7 lines) limits driver-vs-passenger discrimination; authors flag ABHD15, FAM193B, UTRN, GIT2 as expressed mutated candidates needing follow-up.
  • Drug-response data come from a single AR-amplified line (MSK-PCa2); generalizability of the enzalutamide / PI3K-inhibitor combination across CRPC subtypes is unproven.
  • No formal comparison to standard 2D prostate cell lines (LNCaP, VCaP, etc.) in head-to-head drug screens.

Citations from this paper used in the wiki

  • “Using a 3D ‘organoid’ system, we report success in long-term culture of prostate cancer from biopsy specimens and circulating tumor cells. The first seven fully characterized organoid lines recapitulate the molecular diversity of prostate cancer subtypes, including TMPRSS2-ERG fusion, SPOP mutation, SPINK1 overexpression and CHD1 loss.” (Summary)
  • “The number somatic non-synonymous single nucleotide variations (SNV) and indels ranged from 29 in MSK-PCa1 to 75 in MSK-PCa4 with a mean of 45.4 per sample, consistent with reported mutation frequency in CRPC tissue.” (Results)
  • “MSK-PCa7 harbors a heterozygous SPOP F133L mutation within the substrate recognition domain (a known hotspot in human samples), making it the only known in vitro model of this mutation reported to date.” (Results)
  • “The AR amplified MSK-PCa2 line was exquisitely sensitive to enzalutamide with an IC-50 of approximately 50 nM whereas the other lines were resistant. The MSK-PCa2 organoid line, which harbors both PTEN loss and PIK3R1 mutation, was sensitive to both everolimus and BKM-120.” (Results)
  • “Genetics data from array-CGH, RNA-Seq, and whole-exome sequencing can be analyzed and downloaded from the MSKCC cBioportal (http://www.cbioportal.org).” (Repositories)

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