We report that tumor cells without mitochondrial DNA (mtDNA) show delayed tumor growth, and that tumor formation is associated with acquisition of mtDNA from host cells. This leads to partial recovery of mitochondrial function in cells derived from primary tumors grown from cells without mtDNA and a shorter lag in tumor growth. Cell lines from circulating tumor cells showed further recovery of mitochondrial respiration and an intermediate lag to tumor growth, while cells from lung metastases exhibited full restoration of respiratory function and no lag in tumor growth. Stepwise assembly of mitochondrial respiratory (super)complexes was correlated with acquisition of respiratory function. Our findings indicate horizontal transfer of mtDNA from host cells in the tumor microenvironment to tumor cells with compromised respiratory function to re-establish respiration and tumor-initiating efficacy. These results suggest pathophysiological processes for overcoming mtDNA damage and support the notion of high plasticity of malignant cells.

We have developed two stable murine tumor cell models lacking mtDNA and investigated whether these cells form tumors in syngeneic mice. Our results show delayed tumor growth and acquisition of mtDNA from host cells. Cells derived from primary tumors that grow from ρ 0 cells and from their circulating and metastatic counterparts progressively recovered mitochondrial respiratory function, and this was associated with assembly of mitochondrial SCs and correlated with tumorigenicity.

Due to the central role of mitochondria in homeostasis, cells have conserved control systems to maintain mitochondrial integrity, appropriate mitochondrial mass, and bioenergetic and metabolic functions. In many pathologies, mtDNA mutations affect tissues with high demand for energy (). Cancer is characterized by altered energy metabolism () involving not only genetic alterations in nDNA, but also mtDNA mutations and changes in mtDNA copy number (). Certain mtDNA mutations compromise ETC function and contribute to a shift to aerobic glycolysis that typifies metastatic progression (). Relevant studies are limited by the difficulty of experimentally manipulating mtDNA of tumor cells and the paucity of tumor models with mtDNA mutations. A model of severe mtDNA damage involves mitochondrial genome deletion (). These ρcells grow in culture with uridine and pyruvate supplementation, but the question of whether they can form tumors following autologous transplantation has not been studied.

Mitochondria are structurally dynamic organelles with a circular genome (mtDNA) of ∼16.5 kb in mammals that encodes 13 subunits of the respiratory complexes (RCs), 22 tRNAs, and 2 rRNAs. Cells contain both discreet and networked mitochondria each with multiple mtDNA copies. Replication and transcription of mtDNA involves the regulatory D-LOOP region, which binds the nuclear-encoded mitochondrial transcription factor TFAM and other regulatory proteins (). The mitochondrial proteome comprises ∼1,500 proteins, most of which are encoded by nuclear DNA (nDNA) (). A major role of mitochondria, ATP generation, is facilitated by oxidative phosphorylation (OXPHOS) comprising four complexes of the electron transport chain (ETC), complex I (CI), CII, CIII and CIV, and CV with ATP synthase activity. RCs form supercomplexes (SCs), such as the respirasome () that transfers electrons from CI to CIV via CIII (). RCs and SCs are assembled stepwise in a process involving specific factors ().

Native blue gel electrophoresis (NBGE) using a mild detergent followed by WB was used to assess the assembly of individual RCs and SCs ( Figure 7 F). Parental cells showed a high level of the fully assembled respirasome (CI+CIII+CIV), which was absent in 4T1ρcells. Lower respirasome levels were observed in 4T1ρSC cells and 4T1ρCTC cells, with full restoration in 4T1ρSCL cells. Free CI, CIII in the form of a dimer and in an SC with CIV, and CIV largely in the form of a monomer, were present at similar levels in all cell types except for 4T1ρcells. CV was partially assembled in 4T1ρcells due to the absence of structural mtDNA-encoded ATP6 and ATP8, while it was fully assembled and present as a dimer in all other cell types. CII was represented in 4T1ρcells by the highly expressed SDHA subunit. SDHB, only observed in fully assembled CII, was lower in 4T1ρ, 4T1ρSC, and 4T1ρCTC than in 4T1 cells and elevated by 70% in 4T1ρSCL cells. These findings demonstrate that transition from 4T1ρCTC to 4T1ρSCL cells yields a fully assembled respirasome and CII, associated with efficient respiration and tumor initiation.

The expression of RC subunit proteins encoded by mtDNA and nDNA, as well as proteins with a regulatory or general role in mitochondria, was investigated in 4T1ρ-derived cell lines and control cells ( Figures 7 E and S3 L). Nuclear-encoded CI subunits NDUFS3 and NDUFA9 were not expressed in 4T1ρcells, but were expressed in all other cell lines. The CII subunit SDHA was highly expressed in all cell lines, while SDHB was expressed at lower levels in 4T1ρ, 4T1ρSC, and 4T1ρCTC cells. The assembly factor SDHAF2 that flavinates SDHA was expressed in all cell lines, but was detected at higher levels in 4T1ρSC cells, while the nuclear-encoded CIII protein Core2 was expressed in all sublines and was lowest in 4T1ρcells. Nuclear-encoded CV ATPaseβ was expressed at similar levels in all sublines. The mtDNA-encoded COI and nDNA-encoded COIV subunits were not expressed in 4T1ρcells, but COI was expressed highly in 4T1ρSC cells. Nuclear-encoded proteins that are not part of RCs were expressed in all sublines with Cyt c being particularly high in 4T1ρcells, and TIM23 and VDAC1 attenuated in these cells. PGCα/β that regulates the transcription factors NRF1/NRF2 was weakly expressed in 4T1ρcells. OPA1, which modulates cristae formation and geometry, was also low in 4T1ρand 4T1ρSC cells, but progressively increased in 4T1ρCTC and 4T1ρSCL cells and was highly expressed in matching 4T1SC and 4T1SCL cells ( Figure S3 L).

We next studied translation of mtDNA-encoded proteins. Figure 7 A shows only background staining in 4T1ρcells, with the proteins coordinately expressed 2- to 3-fold higher in the other sublines compared to parental cells. Activity of citrate synthase (CS), the first enzyme of the TCA cycle, was increased 2.5-fold in 4T1ρand 4T1ρSC cells, while in 4T1ρCTC and 4T1ρSCL cells the activity was similar to that in parental cells ( Figure 7 B). This increase was not observed in the corresponding sublines ( Figure S3 I). We did not see a major difference in succinate dehydrogenase (SDH) activity in sublines derived from 4T1ρcells ( Figure 7 C) or the matching cells ( Figure S3 J). Succinate quinone reductase (SQR) activity of CII was very low in 4T1ρ, 4T1ρSC, and 4T1ρCTC cells, while in 4T1ρSCL cells it was 70% higher than in 4T1 cells ( Figure 7 D). SQR activity was about 2-fold higher in the metastatic 4T1SCL cells than in 4T1 and 4T1SC cells ( Figure S3 K).

(A) 4T1 cells and derived sublines were evaluated for newly synthesized proteins using the Clik-iT AHA kit. Sublines were assessed for CS (B), SDH (C), and SQR activity (D) as detailed in Experimental Procedures , as well as for expression of specific proteins using WB (E). Tubulin was used as a loading control. (F) The mitochondrial fraction was lysed in the presence of digitonin and subjected to NBGE. Specific subunits of individual complexes were detected using the antibodies shown. HSP60 was used as a loading control. The level of expression of the individual bands representing RCs, subcomplexes, and SCs was evaluated by densitometry relative to HSP60. Data are from three independent experiments, and the results are expressed as mean ± SD. The symboldenotes significant differences with p < 0.05.

nDNA-encoded subunits of mitochondrial complexes, with the exception of Sdhc, showed a similar expression pattern indicating a common transcription mechanism ( Figure 6 C). Of the regulatory genes, relative expression of mRNA for the mitochondrial transcription factor Tfam was very low in 4T1ρand almost 3-fold higher in 4T1ρSC cells, but was detected at similar levels in 4T1, 4T1ρCTC, and 4T1ρSCL cells ( Figure 6 D). The Tfam transcript profile in the sublines mirrors the mtDNA/nDNA ratios, suggesting a causal relationship. Nrf1, Nrf2, Tfb1m, Tfb2m, and Top1m transcripts were depressed in 4T1ρSC cells, while the assembly factor Scafi (Cox7a2l) was decreased in 4T1ρ, 4T1ρSC, and 4T1ρCTC cells, and the assembly factor Hig2a was relatively unchanged. No differences in these transcripts were observed in the matching cells ( Figure S3 H).

To determine whether phenotypic differences in 4T1ρ-derived sublines following mtDNA acquisition were related to gradual restoration of mtDNA, we evaluated the mtDNA/nDNA ratio. While 4T1ρcells did not contain mtDNA, we found a several-fold increase in the mtDNA/nDNA ratio in 4T1ρSC compared to parental cells. This ratio was maintained over several months of cell culture ( Figure 6 A). The notion of no mtDNA is corroborated by no product in both B16ρand 4T1ρcells when amplifying whole cell mtDNA for NGS ( Figure S6 ). 4T1ρCTC and 4T1ρSCL cells showed mtDNA/nDNA ratios more similar to those of parental cells as did the corresponding 4T1SC and 4T1SCL cells ( Figure S3 G). Transcripts of mtDNA-encoded genes were not detected in 4T1ρcells, but were found in 4T1ρ-derived sublines. Relative to 4T1 cells, Nd1, Nd2, Nd4, Nd4l, Nd5, Nd6, and mt-Co2 were elevated 2- to 4-fold in 4T1ρSC cells, and Nd1, Nd2, mt-Co2, Atp6, and Atp8 elevated 1.5- to 4.5-fold in 4T1ρSCL cells. The level of D-loop transcript was similar in all 4T1ρ-derived sublines, indicating that the regulatory region of mtDNA does not hinder mtDNA replication or transcription in these cells ( Figure 6 B).

(A) 4T1 cells and derived sublines were evaluated for mtDNA/nDNA ratio using qPCR and specific primers ( Supplemental Information ). Analyses were performed using cells maintained in culture for a short period of time (first analysis), 1 month (second analysis), and 4 months (third analysis). Sublines were also evaluated for the level of RC transcripts encoded by mtDNA (B) and nDNA (C) and for transcripts of mitochondrial regulatory genes (D) using qPCR and specific primers ( Supplemental Information ). Data are from three independent experiments, and the results are expressed as mean ± SD. The symboldenotes significant differences with p < 0.05.

4T1ρcells showed barely detectable respiration, which was low in 4T1ρSC cells and higher in 4T1ρCTC cells, while 4T1ρSCL cells respired at a similar rate to parental cells ( Figures 5 G and S5 A). Respiration was similar in 4T1 and 4T1SC cells and slightly lower in 4T1SCL cells ( Figure S3 F). Respiration via CI and CII was very low in 4T1ρSC cells and higher in 4T1ρCTC cells. Respiration via CI was similar in parental cells and 4T1ρSCL, while respiration via CII was highly elevated in metastatic cells ( Figures 5 H and S5 B). A similar respiration profile was found for isolated mitochondria ( Figure S5 C), indicating that respiration of the sublines is primarily governed by mitochondria, and not the extramitochondrial compartment. Collectively, these results show significant differences in respiration of individual 4T1ρ-derived sublines, which paralleled the efficacy of these cells to initiate tumor growth ( Figure 1 C).

Cells devoid of mtDNA use glycolysis as a source of ATP. Compared to parental 4T1 cells, 4T1ρ, 4T1ρSC, and 4T1ρCTC cells showed elevated glucose uptake, increased production of lactate, and lower levels of ATP ( Figures 5 A–5C). In contrast, 4T1ρSCL cells were less glycolytic and more similar to parental 4T1 cells, as were the matching sublines ( Figures S3 A–S3C). Cellular bioenergetic status and the efficiency of mitochondrial respiration modulate production of reactive oxygen species (ROS). We used the mitochondrially targeted probe, MitoSOX, as an indicator of mitochondrial superoxide generation. Cell lines from primary tumors that developed from 4T1ρcells when injected s.c. or into the mammary fat pad showed distinct intermediate and high fluorescence intensity peaks, while cells from lung metastases showed predominantly a high fluorescence intensity peak comparable with parental 4T1 cells ( Figure S4 A). Only the intermediate peak was present in 4T1ρcells. Similar results were obtained with B16 cells ( Figure S4 B). Quantitation of MitoSOX staining showed low fluorescence in 4T1ρcells with other sublines being comparable with parental 4T1 cells ( Figure 5 D). Dihydrodichlorofluorescein (DCF) measurement of cytosolic hydrogen peroxide showed a nonsignificant decrease in the signal in 4T1ρ, 4T1ρSC, and 4T1ρSCL cells relative to parental 4T1 cells ( Figure 5 E). Using TMRM as a probe, mitochondrial membrane potential (ΔΨ) was found to be similar in 4T1 and 4T1ρcells, elevated in 4T1ρSCL and to a lesser extent in 4T1ρSC cells, and was greatly reduced in 4T1ρCTC cells ( Figure 5 F).

4T1 cells and derived sublines were assessed for glucose uptake (A), lactate production (B), and ATP level (C). Mitochondrial superoxide generation was evaluated using MitoSOX (D), cytosolic hydrogen peroxide with DCF (E), and ΔΨ m using TMRM (F). (G) Cells were evaluated for routine, leak, net (R–L), and uncoupled (ETS) respiration using an Oxygraph respirometer. (H) Cells were permeabilized with digitonin and evaluated for respiration at the presence of substrates specific for CI and CII. Data are from three independent experiments, and the results are expressed as mean ± SD. The symbol ∗ denotes significant differences with p < 0.05.

Investigation of mitochondrial morphology by transmission electron microscopy (TEM) revealed that mitochondria of 4T1ρcells were distended with low-density internal staining and were largely free of stacked cristae, but contained ring-like cristae ( Figure 4 ). Intriguingly, 4T1ρSC cells showed mitochondria with and without cristae, while 4T1ρCTC cells contained more mitochondria with cristae. 4T1ρSCL cells were indistinguishable from parental 4T1 cells and the matching tumor cells ( Figures 4 and S2 B).

4T1 cells and derived sublines were grown on coverslips, processed for TEM, and examined using TEM. The lower graphs indicate percent mitochondria with visible cristae (left) and percent mitochondria with ring-like cristae (right) for each subline. The percentage of mitochondria with visible cristae in 4T1 cells was set as 100%, the percentage of mitochondria with ring-like cristae in 4T1ρ 0 cells set as 100%. Data are from three independent experiments, and the results are expressed as mean ± SD. The symbol ∗ denotes significant differences with p < 0.05.

We tested individual sublines for their autophagic status, reasoning that 4T1ρSC cells may exhibit higher mitophagy to remove respiration-defective mitochondria devoid of mtDNA. Initial acridine orange (AO) staining showed heterogeneous acidic vesicles in 4T1ρSC cells, and western blotting (WB) revealed lower levels of the autophagy marker LC3B-II in 4T1ρand 4T1ρSC cells than in 4T1ρCTC and 4T1ρSCL cells, and higher levels of p62 in 4T1ρSC cells, both with control cells and cells treated with the autophagy inhibitor bafilomycin A1 ( Figure 3 C). The mitophagy marker, Parkin, showed increased expression in cells derived from 4T1ρand highest expression in 4T1ρSCL cells, but BNIP3 expression remained unaltered in the sublines. Since individual sublines were derived from different stages of metastatic tumor progression, we investigated markers of epithelial-mesenchymal transition (EMT). Figure 3 D shows that the sublines differed coordinately in the level of expression of E-cadherin and vimentin consistent with their different origin.

Acquisition of host mtDNA by ρtumor cells prompted us to investigate the phenotypic and morphological properties of the 4T1ρ-derived sublines (4T1ρSC, 4T1ρCTC, and 4T1ρSCL cells). We also investigated “matching” cells derived from s.c. tumors (4T1SC cells) and lung metastases (4T1SCL cells) that grew from parental 4T1 cells. The doubling times of these cell lines were as follows: 4T1, 18.7 hr; 4T1SC, 17.5 hr; 4T1SCL, 16 hr; 4T1ρ, 33.6 hr; 4T1ρSC, 21.5 hr; 4T1ρCTC, 23.1 hr; and 4T1ρSCL, 16.1 hr. Light microscopy revealed morphological differences of individual sublines ( Figure 3 A). In agreement with the PCR analysis of mtDNA ( Figure 2 A), staining with EtBr confirmed the absence of mtDNA in 4T1ρcells, showing only nucleolar staining. Costaining with MitoTracker Red showed high mtDNA heterogeneity in 4T1ρSC and to a lesser extent in 4T1ρCTC cells. Flow cytometric analysis revealed that ∼60% of 4T1ρSC cells contained little or no mtDNA, while almost 90% of 4T1ρCTC and 100% of 4T1ρSCL cells contained mtDNA ( Figure 3 B). 4T1SC cells showed similar mtDNA staining to that found in 4T1 and 4T1SCL cells ( Figure S2 A).

(C) Sublines were incubated with AO (red) to visualize acidic vacuoles and with Hoechst 33342 to show nuclei (blue). Cells were also probed by WB for LC3B-I/II, p62, Parkin, and BNIP3 with actin as loading control. Bafilomycin A treatment (20 nM, 16 hr) was included in the LC3BII analysis to determine autophagic flux.

(B) Sublines were stained with EtBr and MitoTracker Red, and confocal microscopy was used to identify yellow (EtBr) and red (MitoTracker Red) fluorescence. Nuclei were stained with Hoechst 33342 (blue). The percentage of cells with (upper-right quadrant) and without (left quadrants) mtDNA was evaluated by flow cytometry.

PCR analysis of the mtDNA-encoded Cytb gene revealed no product in B16ρor 4T1ρcells confirming the absence of mtDNA in these cells ( Figure 2 A). These results rule out possible contribution from nuclear mitochondrial DNA sequences (). However, Cytb was present in B16ρSC and B16ρSCL cells. Similar results were obtained with 4T1ρSC and 4T1ρSCL cells ( Figure 2 A). To investigate the origin of the mtDNA in these cells, we sequenced the mtDNA region containing the polymorphic tRNAlocus. Cells from both B16ρ- and 4T1ρ-derived tumors showed the polymorphism of the host mouse (), rather than that of B16 and 4T1 cells ( Figure 2 B), demonstrating acquisition of mtDNA from host cells in the tumor microenvironment (eight consecutive adenines for C57BL/6 and nine for NOD/scid and Balb/c mice) to ρtumor cells in each model. To verify the host origin of mtDNA, we sequenced whole mtDNA from B16, B16ρSC, 4T1, and 4T1ρSC cells using next-generation Ion Torrent and Nanopore MinION methodologies. These approaches identified two additional polymorphisms in 16SrRNA (site 1,576) and D-loop (site 16,076) that distinguished 4T1 from 4T1ρSC cells ( Table S1 ), verifying that mtDNA in tumors derived from 4T1ρcells was from the recipient mouse. No additional polymorphisms were observed in B16 and B16ρSC cells. Taken together, these findings unequivocally demonstrate that mtDNA in tumors derived from ρcells originate from the recipient mouse. Mitochondrial protein synthesis was shown to be functional in cell lines derived from tumors from B16ρand 4T1ρcells by the presence of COI, an mtDNA-encoded protein ( Figure 2 C).

(B) Left panels: sequence analysis of the polymorphic region (9,817–9,836 C57BL/6 reference NC_005089.1) of the mitochondrial tRNA Arg gene from C57BL/6 and NOD/scid mice and from s.c. tumors derived from B16ρ o cells grown in C57BL/6 and NOD/scid mice. Right panels: comparable sequence analysis of the mitochondrial tRNA Arg gene from Balb/c mice and from a s.c. tumor derived from 4T1ρ 0 cells grown in Balb/c mice. A summary table is presented.

(A) Left panels: Cytb PCR analysis of B16, B16ρ 0 , and B16ρ 0 SC and B16ρ 0 SCL tumor sublines from C57BL/6 (upper-left panel) and NOD/scid mice (lower-left panel). Right panels: Cytb PCR analysis of 4T1, 4T1ρ 0 , and tumor sublines from orthotopic primary and metastatic tumors (upper-right panel, 4T1ρ 0 MFP, 4T1ρ 0 MFPL) and s.c. primary and metastatic tumors (4T1ρ 0 SC, 4T1ρ 0 SCL) that form in Balb/c mice.

To investigate whether acquisition of tumorigenic properties is associated with respiratory competence, tumors that formed from 4T1, 4T1ρ, and derived cell lines were assessed by high-resolution respirometry. Relative to cells from parental tumors, respiration by tumors that grew from 4T1ρcells was reduced by 60%, whereas 4T1ρSC, 4T1ρCTC, and 4T1ρSCL cell-derived tumors exhibited progressive recovery of respiration ( Figure 1 D). Histological examination of tumors derived from each cell line showed qualitative similarities ( Figure 1 E).

As 4T1 and 4T1ρcells are resistant to 6-thioguanine (6TG), we used 20 μM 6TG to select cell lines from primary tumors that formed from 4T1ρcells (4T1ρSC), circulating tumor cells (4T1ρCTC), and lung metastases (4T1ρSCL). When injected i.v., both 4T1ρSC and 4T1ρSCL cells, and a cell line derived from lung metastases that formed from an orthotopic tumor (4T1ρMFPL), formed lung tumors that were comparable to 4T1 cells ( Figure 1 B). When injected s.c. into Balb/c mice, 4T1ρSC cells formed tumors with a delay of ∼10 days, whereas 4T1ρSCL cells initiated tumors without a lag ( Figure 1 C; n = 2 experiments). 4T1ρCTC cells formed tumors with a lag between that of 4T1ρSC and 4T1ρSCL cells.

To model severe mitochondrial genome damage, we deleted mtDNA in metastatic murine B16 melanoma and 4T1 breast carcinoma cells using low-dose ethidium bromide (EtBr), thereby generating B16ρand 4T1ρcells that have been maintained as stable auxotrophic cell lines without EtBr for more than 2 years. When injected subcutaneously (s.c.) into groups of five syngeneic C57BL/6 (n = 3 experiments) or NOD/scid (n = 3 experiments) mice, B16ρcells (10) initiated tumors with a delay of more than 20 days compared to parental cells ( Figure 1 A). Intravenous (i.v.) injection of B16ρcells into NOD/scid mice failed to generate lung tumors. However, cell lines derived from s.c. tumors and lung metastases that grew from B16ρcells following long-term culture formed lung tumors at a similar frequency to B16 cells ( Figure 1 A). These cell lines were shown to be of B16, rather than mouse origin, because they produced melanin under stress. Similarly, 4T1ρcells formed tumors with a lag of 20–25 days when 10cells were injected s.c. (n = 6 experiments) or orthotopically into the mammary fat pad (n = 1 experiment) of Balb/c mice, but failed to form lung tumors following i.v. injection ( Figure 1 B) or following s.c. injection of ≤ 10cells ( Figure S1 available online).

(E) Histological staining of tissue sections from (C) stained with H&E; scale bar, 20 μm. Data are from three independent experiments, and the results are expressed as mean ± SD. The symbol ∗ denotes significant differences with p < 0.05 and ∗∗ p < 0.001.

(D) Oxygen consumption by tissue of tumors derived from cells as stated above (C) was compared with respiration of liver tissue obtained from the same mice.

(B) 4T1 and 4T1ρ 0 cells were injected s.c. or orthotopically into the mammary fat pad of Balb/c mice, and tumor growth was monitored. These cells and 6TG-resistant cell lines derived from s.c. tumors and lung metastases that grew from 4T1ρ 0 cells were also injected i.v. into Balb/c mice, and lung weight and tumor colonies were determined. Images of lungs from these mice are also shown.

(A) B16 and B16ρ 0 cells (10 5 ) were injected s.c. into C57BL/6 or NOD/scid mice, and tumor growth was monitored. These cells and cell lines derived from s.c. tumors and lung metastases that grew from B16ρ 0 cells were also injected i.v. into NOD/scid mice, and lung weight and tumor colonies were determined.

Discussion

0 melanoma and 4T1ρ0 breast carcinoma cells in C57BL/6 and Balb/c mice, respectively, as well as in NOD/scid mice. Previous reports on grafting human ρ0 tumor cells have provided variable results, depending on tumor type, the site of injection, and the recipient mouse ( Hayashi et al., 1992 Hayashi J.

Takemitsu M.

Nonaka I. Recovery of the missing tumorigenicity in mitochondrial DNA-less HeLa cells by introduction of mitochondrial DNA from normal human cells. Morais et al., 1994 Morais R.

Zinkewich-Péotti K.

Parent M.

Wang H.

Babai F.

Zollinger M. Tumor-forming ability in athymic nude mice of human cell lines devoid of mitochondrial DNA. Cavalli et al., 1997 Cavalli L.R.

Varella-Garcia M.

Liang B.C. Diminished tumorigenic phenotype after depletion of mitochondrial DNA. Magda et al., 2008 Magda D.

Lecane P.

Prescott J.

Thiemann P.

Ma X.

Dranchak P.K.

Toleno D.M.

Ramaswamy K.

Siegmund K.D.

Hacia J.G. mtDNA depletion confers specific gene expression profiles in human cells grown in culture and in xenograft. Kulawiec et al., 2008 Kulawiec M.

Safina A.

Desouki M.M.

Still I.

Matsui S.

Bakin A.

Singh K.K. Tumorigenic transformation of human breast epithelial cells induced by mitochondrial DNA depletion. Imanishi et al., 2011 Imanishi H.

Hattori K.

Wada R.

Ishikawa K.

Fukuda S.

Takenaga K.

Nakada K.

Hayashi J. Mitochondrial DNA mutations regulate metastasis of human breast cancer cells. 0 and 4T1ρ0 cells were found to be positive for both the Cytb gene and COI protein. We excluded the possibility of latent mtDNA being present in ρ0 cells by showing that tumors growing from B16ρ0 and 4T1ρ0 cells contained the tRNAArg gene polymorphism of the recipient mouse ( Bayona-Bafaluy et al., 2003 Bayona-Bafaluy M.P.

Acín-Pérez R.

Mullikin J.C.

Park J.S.

Moreno-Loshuertos R.

Hu P.

Pérez-Martos A.

Fernández-Silva P.

Bai Y.

Enríquez J.A. Revisiting the mouse mitochondrial DNA sequence. 0SC cells and the recipient mouse. Although our results cannot formally disprove the possibility of residual latent mtDNA being present in ρ0 cells, and that low-level heteroplasmy might occur at any particular polymorphic site in this latent mtDNA, the probability that latency and homoplastic conversion at all three sites could explain our results is considered to be exceedingly remote. Using metastatic murine tumor models with mtDNA deletion to simulate severe mtDNA damage, we have found a long lag to tumor formation for both B16ρmelanoma and 4T1ρbreast carcinoma cells in C57BL/6 and Balb/c mice, respectively, as well as in NOD/scid mice. Previous reports on grafting human ρtumor cells have provided variable results, depending on tumor type, the site of injection, and the recipient mouse (). In both models, we observed delayed tumor growth that was associated with mtDNA acquisition from the host suggestive of horizontal mtDNA transfer. This was demonstrated when cells derived from tumors that grew from B16ρand 4T1ρcells were found to be positive for both the Cytb gene and COI protein. We excluded the possibility of latent mtDNA being present in ρcells by showing that tumors growing from B16ρand 4T1ρcells contained the tRNAgene polymorphism of the recipient mouse (), which is distinct from that of the original tumor. Two additional 4T1 polymorphisms, D-loop and 16S rRNA, were identical in 4T1ρSC cells and the recipient mouse. Although our results cannot formally disprove the possibility of residual latent mtDNA being present in ρcells, and that low-level heteroplasmy might occur at any particular polymorphic site in this latent mtDNA, the probability that latency and homoplastic conversion at all three sites could explain our results is considered to be exceedingly remote.

To better understand the role of mitochondrial respiration in tumorigenesis and metastasis, we characterized malignant cells from different tumor microenvironments in the 4T1ρ0 model. To ensure that the results relate to mtDNA acquisition and not local microenvironment, we isolated matching cell lines from primary s.c. 4T1 tumors and lung metastases. Interestingly, we found that cells derived from primary tumors that grew from 4T1ρ0 cells still exhibited a considerable delay in tumor initiation, while the kinetics of growth of tumors derived from the metastatic 4T1ρ0SCL cells showed no delay. These findings, initially acquired in our laboratory in Wellington and repeated with almost identical results in our laboratory in Prague 9 months later, indicate the robust nature of these results and high reproducibility.

0 cells to low levels in primary tumor cells derived from ρ0 cells, progressing to higher levels of respiration in CTCs and full restoration in metastatic cells. The phenotype of the individual cell types was maintained in culture, as documented by sustained high mtDNA/nDNA ratio in cells from the primary tumor. This suggests that these cells are unable to restore full mitochondrial function during long-term maintenance in vitro and need microenvironmental exposure to progress to full respiratory competence. Our findings are consistent with earlier in vitro results ( Spees et al., 2006 Spees J.L.

Olson S.D.

Whitney M.J.

Prockop D.J. Mitochondrial transfer between cells can rescue aerobic respiration. 0 lung adenocarcinoma cells restored their respiratory capacity. A striking finding is the stepwise recovery of mitochondrial respiration from undetectable levels in ρcells to low levels in primary tumor cells derived from ρcells, progressing to higher levels of respiration in CTCs and full restoration in metastatic cells. The phenotype of the individual cell types was maintained in culture, as documented by sustained high mtDNA/nDNA ratio in cells from the primary tumor. This suggests that these cells are unable to restore full mitochondrial function during long-term maintenance in vitro and need microenvironmental exposure to progress to full respiratory competence. Our findings are consistent with earlier in vitro results () showing that mitochondrial transfer from MSCs and skin fibroblasts to human A549ρlung adenocarcinoma cells restored their respiratory capacity.

LeBleu et al., 2014 LeBleu V.S.

O’Connell J.T.

Gonzalez Herrera K.N.

Wikman H.

Pantel K.

Haigis M.C.

de Carvalho F.M.

Damascena A.

Domingos Chinen L.T.

Rocha R.M.

et al. PGC-1α mediates mitochondrial biogenesis and oxidative phosphorylation in cancer cells to promote metastasis. Lapuente-Brun et al., 2013 Lapuente-Brun E.

Moreno-Loshuertos R.

Acín-Pérez R.

Latorre-Pellicer A.

Colás C.

Balsa E.

Perales-Clemente E.

Quirós P.M.

Calvo E.

Rodríguez-Hernández M.A.

et al. Supercomplex assembly determines electron flux in the mitochondrial electron transport chain. Moreno-Lastres et al., 2012 Moreno-Lastres D.

Fontanesi F.

García-Consuegra I.

Martín M.A.

Arenas J.

Barrientos A.

Ugalde C. Mitochondrial complex I plays an essential role in human respirasome assembly. 0 cells. The key regulatory role of SCAFI and other respirasome assembly factors in the tumor microenvironment in metastatic progression needs further investigation. Similarly, the reasons for full assembly of CII in metastatic cells are not known, but are likely linked to retrograde mitochondrial signaling. That the assembly of RCs and SCs is crucial for the full recovery of mitochondrial respiration is demonstrated by our findings that except for ρ0 cells, all sublines of these cells exhibited transcriptional and translational activity of mtDNA-encoded genes, with partially or fully assembled CI, CIII, and CIV. A key to understanding the different behavior of the sublines may relate to the high level of mitochondrial heterogeneity. We show detailed analysis of the individual stages of recovery of respiratory competence from parental cells to their metastatic counterparts, documenting each “recovery” stage. A crucial step of full respiration recovery is associated with the assembly of the respirasome and CII, demonstrating the need for fully functional OXPHOS. This is also supported by the high level of SQR activity in metastatic cells and agrees with the requirement for efficient OXPHOS in metastatic dissemination (). In addition, we show that the level of the assembly factor SCAFI (), but not HIG2A (), correlates with the extent of respirasome assembly in tumors derived from 4T1ρcells. The key regulatory role of SCAFI and other respirasome assembly factors in the tumor microenvironment in metastatic progression needs further investigation. Similarly, the reasons for full assembly of CII in metastatic cells are not known, but are likely linked to retrograde mitochondrial signaling. That the assembly of RCs and SCs is crucial for the full recovery of mitochondrial respiration is demonstrated by our findings that except for ρcells, all sublines of these cells exhibited transcriptional and translational activity of mtDNA-encoded genes, with partially or fully assembled CI, CIII, and CIV. A key to understanding the different behavior of the sublines may relate to the high level of mitochondrial heterogeneity.

0 cells, having acquired mtDNA, progress from primary tumor via the circulation to lung metastases, and how this progression leads to full recovery of respiration. In our model, metastatic potential is encoded in the nuclear genome, but suppressed by the absence of mtDNA. Acquisition of “normal” mtDNA from the microenvironment enables 4T1ρ0 cells to grow as primary tumors and “unleashes” their metastatic potential. It is possible that rare cells acquire a few mitochondria that contain mtDNA, and that these cells replicate their mtDNA, build cisternal structures of cristae, and multiply clonally at the expense of cells lacking mtDNA that are progressively eliminated by autophagy. Our finding that autophagic activity is low in 4T1ρ0 and 4T1ρ0SC cells that also feature lower respiration is consistent with a report showing faulty autophagy in cells with suppressed ETC ( Graef and Nunnari, 2011 Graef M.

Nunnari J. Mitochondria regulate autophagy by conserved signalling pathways. A fundamental question raised by our findings relates to how ρcells, having acquired mtDNA, progress from primary tumor via the circulation to lung metastases, and how this progression leads to full recovery of respiration. In our model, metastatic potential is encoded in the nuclear genome, but suppressed by the absence of mtDNA. Acquisition of “normal” mtDNA from the microenvironment enables 4T1ρcells to grow as primary tumors and “unleashes” their metastatic potential. It is possible that rare cells acquire a few mitochondria that contain mtDNA, and that these cells replicate their mtDNA, build cisternal structures of cristae, and multiply clonally at the expense of cells lacking mtDNA that are progressively eliminated by autophagy. Our finding that autophagic activity is low in 4T1ρand 4T1ρSC cells that also feature lower respiration is consistent with a report showing faulty autophagy in cells with suppressed ETC (). Such cells do not appear to “clear” dysfunctional mitochondria, which is more likely to occur at the stage of CTCs when respiration is partially restored and autophagy higher.

0SC cells, consistent with high levels of TFAM, a factor critical for replication, transcription, and packaging of mtDNA ( Kukat and Larsson, 2013 Kukat C.

Larsson N.G. mtDNA makes a U-turn for the mitochondrial nucleoid. 0 cells and poorly formed cristae in many 4T1ρ0SC cells, consistent with low OPA1 levels ( Cogliati et al., 2013 Cogliati S.

Frezza C.

Soriano M.E.

Varanita T.

Quintana-Cabrera R.

Corrado M.

Cipolat S.

Costa V.

Casarin A.

Gomes L.C.

et al. Mitochondrial cristae shape determines respiratory chain supercomplexes assembly and respiratory efficiency. 0 cells that have acquired mtDNA require conditioning by the local microenvironment to restore a parental intracellular distribution of mtDNA might relate to progressive changes in mitochondria-to-nucleus retrograde signaling that eventually normalizes both mtDNA distribution and respiratory function. The importance of the tumor microenvironment is corroborated by the finding that 4T1 cells show both epithelial and mesenchymal markers, highly localized within the cell and exhibiting different patterns of compartmentalization, with coordinate changes in E-cadherin and vimentin staining across 4T1 cells and derived sublines consistent with the different origins of the individual 4T1 sublines. Quantitation of mtDNA/nDNA ratios showed stable elevation in 4T1ρSC cells, consistent with high levels of TFAM, a factor critical for replication, transcription, and packaging of mtDNA (). Compromised respiration is correlated with lack of cristae in all 4T1ρcells and poorly formed cristae in many 4T1ρSC cells, consistent with low OPA1 levels (). The reasons why ρcells that have acquired mtDNA require conditioning by the local microenvironment to restore a parental intracellular distribution of mtDNA might relate to progressive changes in mitochondria-to-nucleus retrograde signaling that eventually normalizes both mtDNA distribution and respiratory function. The importance of the tumor microenvironment is corroborated by the finding that 4T1 cells show both epithelial and mesenchymal markers, highly localized within the cell and exhibiting different patterns of compartmentalization, with coordinate changes in E-cadherin and vimentin staining across 4T1 cells and derived sublines consistent with the different origins of the individual 4T1 sublines.