Esophageal 3D organoids of MPV17-/- mouse model of mitochondrial DNA depletion show epithelial cell plasticity and telomere attrition

Esophageal squamous cell carcinoma (ESCC) is an aggressive cancer with late-stage detection and poor prognosis. This emphasizes the need to identify new markers for early diagnosis and treatment. Altered mitochondrial genome (mtDNA) content in primary tumors correlates with poor patient prognosis. Here we used three-dimensional (3D) organoids of esophageal epithelial cells (EECs) from the MPV17-/- mouse model of mtDNA depletion to investigate the contribution of reduced mtDNA content in ESCC oncogenicity. To test if mtDNA defects are a contributing factor in ESCC, we used oncogenic stimuli such as ESCC carcinogen 4-nitroquinoline oxide (4-NQO) treatment, or expressing p53R175H oncogenic driver mutation. We observed that EECs and 3D-organoids with mtDNA depletion had cellular, morphological and genetic alterations typical of an oncogenic transition. Furthermore, mitochondrial dysfunction induced cellular transformation is accompanied by elevated mitochondrial fission protein, DRP1 and pharmacologic inhibition of mitochondrial fission by mDivi-1 in the MPV17-/- organoids reversed the phenotype to that of normal EEC organoids. Our studies show that mtDNA copy number depletion, activates a mitochondrial retrograde response, potentiates telomere defects, and increases the oncogenic susceptibility towards ESCC. Furthermore, mtDNA depletion driven cellular plasticity is mediated via altered mitochondrial fission-fusion dynamics.


TFAM KO animals
Tfam expression is allelic copy number dependent (heterogyzous KO have 50% reduced and homogyzous KO have 100% reduced Tfam mRNA compared to wt). Esophageal epithelial cells isolated from TFAM +/fl mice were transduced ex vivo with either adenoCMV or adenoCRE. mtDNA copy number correlates with the Tfam transcript levels.

Tfam floxed esophageal epithelial cells
For ex vivo recombination experiments, esophageal epithelial cells isolated from Tfam +/fl mice were transduced with adenovirus vector containing either CMV or CRE recombinase (University of Iowa Gene Transfer Vector Core). Adenovirus vector containing CMV is used as control. Adenovirus vectors were used at 1:500.

4Nitroquinoline-N-Oxide (4NQO) treatment in vivo
MPV17 -/mice (n =), 7.0 weeks age received 100 µg/ml 4NQO in 2% propylene glycol (MP Biomedicals, Solon, OH) in drinking water ad libitum for 8 weeks (or earlier if mice developed visible signs of discomfort or sickness) and followed for another 4 weeks without treatment. In parallel experiment, littermates were treated with 2% propylene glycol as control group. All experiments and treatments were done under approved protocol from the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC #805731). Both male and female mice were included in this study and animals were randomized to treatment groups.

Histopathological examination
At necropsy, the entire length of the esophagus was dissected longitudinally. The tongue and the esophagus were examined for the presence of macroscopic topographical alterations. The entire esophagus were fixed overnight in 10% neutral buffered formalin, transferred to 70% ethanol and embedded in paraffin. 5 μm thick sections were obtained from the paraffin-embedded blocks and stained with hematoxylin and eosin. Sections were examined for the presence of epithelial atypia, dysplasia and neoplasia using established criteria. Normal histopathology was assessed by examining sections from the wild-type, non-carcinogen treated mice. Subsequent sections were analyzed blinded to the genotype and carcinogen treatment. Depending on the degree of atypical cytological and morphological changes, lesions were scored as mild, moderate or severe hyperplasia and dysplasia.

Mitochondrial membrane potential
Mitochondrial membrane potential in primary EECs derived from TFAM +/fl + adenoCMV and TFAM +/fl + adenoCRE was analyzed using the Cellometer Vision CBA. The MitoProbe™JC-1 (Invitrogen, Carlsbad, CA) assay was used for mitochondrial membrane potential analysis. The assay measures the depolarization of mitochondrial membrane potential. Cells were stained by the JC-1 dye, which exhibits membrane potentialdependent accumulation in mitochondria. The JC-1 fluorescent dye forms red fluorescent J-aggregates (590 nm) when accumulated in the mitochondria. Depolarization of mitochondrial membrane potential was indicated by JC-1 emission shifting from red (590 nm) to green (529 nm) fluorescence (indicated by lower FL2 intensity) due to the loss of concentration-dependent formation of red fluorescent J-aggregates.