Author Correction: Metastatic-niche labelling reveals parenchymal cells with stem features.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Metastatic-niche labelling reveals parenchymal cells with stem features.

Direct investigation of the early cellular changes induced by metastatic cells within the surrounding tissue remains a challenge. Here we present a system in which metastatic cancer cells release a cell-penetrating fluorescent protein, which is taken up by neighbouring cells and enables spatial identification of the local metastatic cellular environment. Using this system, tissue cells with low representation in the metastatic niche can be identified and characterized within the bulk tissue. To highlight its potential, we applied this strategy to study the cellular environment of metastatic breast cancer cells in the lung. We report the presence of cancer-associated parenchymal cells, which exhibit stem-cell-like features, expression of lung progenitor markers, multi-lineage differentiation potential and self-renewal activity. In ex vivo assays, lung epithelial cells acquire a cancer-associated parenchymal-cell-like phenotype when co-cultured with cancer cells and support their growth. These results highlight the potential of this method as a platform for new discoveries.

On monocytes and lymphocytes biolens clustering by in flow holographic microscopy.

Live cells act as biological lenses and can be employed as real-world optical components in bio-hybrid systems. Imaging at nanoscale, optical tweezers, lithography and also photonic waveguiding are some of the already proven functionalities, boosted by the advantage that cells are fully biocompatible for intra-body applications. So far, various cell types have been studied for this purpose, such as red blood cells, bacterial cells, stem cells and yeast cells. White Blood Cells (WBCs) play a very important role in the regulation of the human body activities and are usually monitored for assessing its health. WBCs can be considered bio-lenses but, to the best of our knowledge, characterization of their optical properties have not been investigated yet. Here, we report for the first time an accurate study of two model classes of WBCs (i.e., monocytes and lymphocytes) by means of a digital holographic microscope coupled with a microfluidic system, assuming WBCs bio-lens characteristics. Thus, quantitative phase maps for many WBCs have been retrieved in flow-cytometry (FC) by achieving a significant statistical analysis to prove the enhancement in differentiation among sphere-like bio-lenses according to their sizes (i.e., diameter d) exploiting intensity parameters of the modulated light in proximity of the cell optical axis. We show that the measure of the low intensity area (S: I z < I th z ) in a fixed plane, is a feasible parameter for cell clustering, while achieving robustness against experimental misalignments and allowing to adjust the measurement sensitivity in post-processing. 2D scatterplots of the identified parameters (d-S) show better differentiation respect to the 1D case. The results show that the optical focusing properties of WBCs allow the clustering of the two populations by means of a mere morphological analysis, thus leading to the new concept of cell-optical-fingerprint avoiding fluorescent dyes. This perspective can open new routes in biomedical sciences, such as the chance to find optical-biomarkers at single cell level for label-free diagnosis.

The multifaceted effects of metformin on tumor microenvironment.

The efficacy of metformin in treating cancer has been extensively investigated since epidemiologic studies associated this anti-diabetic drug with a lower risk of cancer incidence. Since tumors are complex systems, in which cancer cells coexist and interact with several different types of non-malignant cells, it is not surprising that anti-cancer drugs affect not only cancer cells, but also the abundance and functions of cells of the tumor microenvironment. Recent years have seen a wide collection of reports showing how metformin, as well as other complex I inhibitors, may influence cancer progression by modulating the phenotype of non-transformed cells in a tumor. In this review, we particularly focus on the effect of metformin on angiogenesis, cancer-associated fibroblasts, tumor-associated macrophages and cancer immunosuppression.

Packaging and transfer of mitochondrial DNA via exosomes regulate escape from dormancy in hormonal therapy-resistant breast cancer.

The horizontal transfer of mtDNA and its role in mediating resistance to therapy and an exit from dormancy have never been investigated. Here we identified the full mitochondrial genome in circulating extracellular vesicles (EVs) from patients with hormonal therapy-resistant (HTR) metastatic breast cancer. We generated xenograft models of HTR metastatic disease characterized by EVs in the peripheral circulation containing mtDNA. Moreover, these human HTR cells had acquired host-derived (murine) mtDNA promoting estrogen receptor-independent oxidative phosphorylation (OXPHOS). Functional studies identified cancer-associated fibroblast (CAF)-derived EVs (from patients and xenograft models) laden with whole genomic mtDNA as a mediator of this phenotype. Specifically, the treatment of hormone therapy (HT)-naive cells or HT-treated metabolically dormant populations with CAF-derived mtDNAhi EVs promoted an escape from metabolic quiescence and HTR disease both in vitro and in vivo. Moreover, this phenotype was associated with the acquisition of EV mtDNA, especially in cancer stem-like cells, expression of EV mtRNA, and restoration of OXPHOS. In summary, we have demonstrated that the horizontal transfer of mtDNA from EVs acts as an oncogenic signal promoting an exit from dormancy of therapy-induced cancer stem-like cells and leading to endocrine therapy resistance in OXPHOS-dependent breast cancer.

Platinum-induced mitochondrial DNA mutations confer lower sensitivity to paclitaxel by impairing tubulin cytoskeletal organization.

Development of chemoresistance is a cogent clinical issue in oncology, whereby combination of anticancer drugs is usually preferred also to enhance efficacy. Paclitaxel (PTX), combined with carboplatin, represents the standard first-line chemotherapy for different types of cancers. We here depict a double-edge role of mitochondrial DNA (mtDNA) mutations induced in cancer cells after treatment with platinum. MtDNA mutations were positively selected by PTX, and they determined a decrease in the mitochondrial respiratory function, as well as in proliferative and tumorigenic potential, in terms of migratory and invasive capacity. Moreover, cells bearing mtDNA mutations lacked filamentous tubulin, the main target of PTX, and failed to reorient the Golgi body upon appropriate stimuli. We also show that the bioenergetic and cytoskeletal phenotype were transferred along with mtDNA mutations in transmitochondrial hybrids, and that this also conferred PTX resistance to recipient cells. Overall, our data show that platinum-induced deleterious mtDNA mutations confer resistance to PTX, and confirm what we previously reported in an ovarian cancer patient treated with carboplatin and PTX who developed a quiescent yet resistant tumor mass harboring mtDNA mutations.

Mild phenotypes and proper supercomplex assembly in human cells carrying the homoplasmic m.15557G > A mutation in cytochrome b gene.

Respiratory complex III (CIII) is the first enzymatic bottleneck of the mitochondrial respiratory chain both in its native dimeric form and in supercomplexes. The mammalian CIII comprises 11 subunits among which cytochrome b is central in the catalytic core, where oxidation of ubiquinol occurs at the Qo site. The Qo- or PEWY-motif of cytochrome b is the most conserved through species. Importantly, the highly conserved glutamate at position 271 (Glu271) has never been studied in higher eukaryotes so far and its role in the Q-cycle remains debated. Here, we showed that the homoplasmic m.15557G > A/MT-CYB, which causes the p.Glu271Lys amino acid substitution predicted to dramatically affect CIII, induces a mild mitochondrial dysfunction in human transmitochondrial cybrids. Indeed, we found that the severity of such mutation is mitigated by the proper assembly of CIII into supercomplexes, which may favor an optimal substrate channeling and buffer superoxide production in vitro.

Pathological and molecular diagnosis of bilateral inguinal lymph nodes metastases from low-grade endometrial adenocarcinoma: a case report with review of the literature.

BACKGROUND: Extra-abdominal metastases in low grade endometrial carcinoma are rare events. Inguinal lymphatic spread occurs usually in advanced disease and is associated with abdominal lymph nodes involvement. To our knowledge, isolated inguinal lymph node metastases in patients with early endometrial carcinoma have never been described thus far. CASE PRESENTATION: We present an uncommon case of inguinal lymph node metastasis in a 51-year old patient with early endometrial disease without other metastatic involvement. The metastatic loci were analyzed with the recently validated method of mitochondrial DNA sequencing to demonstrate clonality of the lesions. CONCLUSIONS: We describe the first case of inguinal metastasis from intramucous endometrial carcinoma; this case confirms the unpredictable spread of endometrial neoplasia and the importance of both patient's history and physical examination in good clinical practice.

Unravelling the Effects of the Mutation m.3571insC/MT-ND1 on Respiratory Complexes Structural Organization.

Mammalian respiratory complex I (CI) biogenesis requires both nuclear and mitochondria-encoded proteins and is mostly organized in respiratory supercomplexes. Among the CI proteins encoded by the mitochondrial DNA, NADH-ubiquinone oxidoreductase chain 1 (ND1) is a core subunit, evolutionary conserved from bacteria to mammals. Recently, ND1 has been recognized as a pivotal subunit in maintaining the structural and functional interaction among the hydrophilic and hydrophobic CI arms. A critical role of human ND1 both in CI biogenesis and in the dynamic organization of supercomplexes has been depicted, although the proof of concept is still missing and the critical amount of ND1 protein necessary for a proper assembly of both CI and supercomplexes is not defined. By exploiting a unique model in which human ND1 is allotopically re-expressed in cells lacking the endogenous protein, we demonstrated that the lack of this protein induces a stall in the multi-step process of CI biogenesis, as well as the alteration of supramolecular organization of respiratory complexes. We also defined a mutation threshold for the m.3571insC truncative mutation in mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1 (MT-ND1), below which CI and its supramolecular organization is recovered, strengthening the notion that a certain amount of human ND1 is required for CI and supercomplexes biogenesis.

Molecular and metabolic features of oncocytomas: Seeking the blueprints of indolent cancers.

Oncocytic tumors are a peculiar subset of human neoplasms in which mitochondria have been proven to have a prominent role. A number of paradoxes render these clinical entities interesting from the translational research point of view. Most oncocytic tumors are generally metabolically constrained due to the impaired respiratory capacity and lack of the ability to respond to hypoxia, yet they maintain features that allow them to strive and persist in an indolent form. Their unique molecular and metabolic characteristics are an object of investigation that may reveal novel ways for therapeutic strategies based on metabolic targeting. With this aim in mind, we here examine the current knowledge on oncocytomas and delve into the molecular causes and consequences that revolve around the oncocytic phenotype, to understand whether we can learn to design therapies from the dissection of benign neoplasms. This article is part of a Special Issue entitled Mitochondria in Cancer, edited by Giuseppe Gasparre, Rodrigue Rossignol and Pierre Sonveaux.

Mitochondrial DNA sequencing demonstrates clonality of peritoneal implants of borderline ovarian tumors.

Borderline ovarian tumors are rare low malignant potential neoplasms characterized by the absence of stromal invasion, whose main prognostic factors are stage and type of peritoneal implants. The latter are defined as invasive when cell proliferation invades the underlying tissue (peritoneal surface, omentum and intestinal wall), or noninvasive. It is still unknown if these implants are metastatic spread from the primary ovarian mass or a neoplastic transformation de novo of the peritoneal surface. Mitochondrial DNA sequencing was performed to assess clonality in eight patients presenting both borderline ovarian tumors and implants. In 37.5% of the cases, the same mitochondrial DNA mutation was present in both borderline ovarian tumors and the peritoneal implant, being this evidence that implants may arise as a consequence of a spread from a single ovarian site.

Different mtDNA mutations modify tumor progression in dependence of the degree of respiratory complex I impairment.

Mitochondrial DNA mutations are currently investigated as modifying factors impinging on tumor growth and aggressiveness, having been found in virtually all cancer types and most commonly affecting genes encoding mitochondrial complex I (CI) subunits. However, it is still unclear whether they exert a pro- or anti-tumorigenic effect. We here analyzed the impact of three homoplasmic mtDNA mutations (m.3460G>A/MT-ND1, m.3571insC/MT-ND1 and m.3243A>G/MT-TL1) on osteosarcoma progression, chosen since they induce different degrees of oxidative phosphorylation impairment. In fact, the m.3460G>A/MT-ND1 mutation caused only a reduction in CI activity, whereas the m.3571insC/MT-ND1 and the m.3243A>G/MT-TL1 mutations induced a severe structural and functional CI alteration. As a consequence, this severe CI dysfunction determined an energetic defect associated with a compensatory increase in glycolytic metabolism and AMP-activated protein kinase activation. Osteosarcoma cells carrying such marked CI impairment displayed a reduced tumorigenic potential both in vitro and in vivo, when compared with cells with mild CI dysfunction, suggesting that mtDNA mutations may display diverse impact on tumorigenic potential depending on the type and severity of the resulting oxidative phosphorylation dysfunction. The modulation of tumor growth was independent from reactive oxygen species production but correlated with hypoxia-inducible factor 1α stabilization, indicating that structural and functional integrity of CI and oxidative phosphorylation are required for hypoxic adaptation and tumor progression.

The cytochrome b p.278Y>C mutation causative of a multisystem disorder enhances superoxide production and alters supramolecular interactions of respiratory chain complexes.

Cytochrome b is the only mtDNA-encoded subunit of the mitochondrial complex III (CIII), the functional bottleneck of the respiratory chain. Previously, the human cytochrome b missense mutation m.15579A>G, which substitutes the Tyr 278 with Cys (p.278Y>C), was identified in a patient with severe exercise intolerance and multisystem manifestations. In this study, we characterized the biochemical properties of cybrids carrying this mutation and report that the homoplasmic p.278Y>C mutation caused a dramatic reduction in the CIII activity and in CIII-driven mitochondrial ATP synthesis. However, the CI, CI + CIII and CII + CIII activities and the rate of ATP synthesis driven by the CI or CII substrate were only partially reduced or unaffected. Consistent with these findings, mutated cybrids maintained the mitochondrial membrane potential in the presence of oligomycin, indicating that it originated from the respiratory electron transport chain. The p.278Y>C mutation enhanced superoxide production, as indicated by direct measurements in mitochondria and by the imbalance of glutathione homeostasis in intact cybrids. Remarkably, although the assembly of CI or CIII was not affected, the examination of respiratory supercomplexes revealed that the amounts of CIII dimer and III2IV1 were reduced, whereas those of I1III2IVn slightly increased. We therefore suggest that the deleterious effects of p.278Y>C mutation on cytochrome b are palliated when CIII is assembled into the supercomplexes I1III2IVn, in contrast to when it is found alone. These findings underline the importance of supramolecular interactions between complexes for maintaining a basal respiratory chain activity and shed light to the molecular basis of disease manifestations associated with this mutation.

Somatic complex I disruptive mitochondrial DNA mutations are modifiers of tumorigenesis that correlate with low genomic instability in pituitary adenomas.

Mitochondrial DNA (mtDNA) mutations leading to the disruption of respiratory complex I (CI) have been shown to exhibit anti-tumorigenic effects, at variance with those impairing only the function but not the assembly of the complex, which appear to contribute positively to cancer development. Owing to the challenges in the analysis of the multi-copy mitochondrial genome, it is yet to be determined whether tumour-associated mtDNA lesions occur as somatic modifying factors or as germ-line predisposing elements. Here we investigated the whole mitochondrial genome sequence of 20 pituitary adenomas with oncocytic phenotype and identified pathogenic and/or novel mtDNA mutations in 60% of the cases. Using highly sensitive techniques, namely fluorescent PCR and allele-specific locked nucleic acid quantitative PCR, we identified the most likely somatic nature of these mutations in our sample set, since none of the mutations was detected in the corresponding blood tissue of the patients analysed. Furthermore, we have subjected a series of 48 pituitary adenomas to a high-resolution array comparative genomic hybridization analysis, which revealed that CI disruptive mutations, and the oncocytic phenotype, significantly correlate with low number of chromosomal aberrations in the nuclear genome. We conclude that CI disruptive mutations in pituitary adenomas are somatic modifiers of tumorigenesis most likely contributing not only to the development of oncocytic change, but also to a less aggressive tumour phenotype, as indicated by a stable karyotype.

A mutation screening of oncogenes, tumor suppressor gene TP53 and nuclear encoded mitochondrial complex I genes in oncocytic thyroid tumors.

BACKGROUND: Thyroid neoplasias with oncocytic features represent a specific phenotype in non-medullary thyroid cancer, reflecting the unique biological phenomenon of mitochondrial hyperplasia in the cytoplasm. Oncocytic thyroid cells are characterized by a prominent eosinophilia (or oxyphilia) caused by mitochondrial abundance. Although disruptive mutations in the mitochondrial DNA (mtDNA) are the most significant hallmark of such tumors, oncocytomas may be envisioned as heterogeneous neoplasms, characterized by multiple nuclear and mitochondrial gene lesions. We investigated the nuclear mutational profile of oncocytic tumors to pinpoint the mutations that may trigger the early oncogenic hit. METHODS: Total DNA was extracted from paraffin-embedded tissues from 45 biopsies of oncocytic tumors. High-resolution melting was used for mutation screening of mitochondrial complex I subunits genes. Specific nuclear rearrangements were investigated by RT-PCR (RET/PTC) or on isolated nuclei by interphase FISH (PAX8/PPARγ). Recurrent point mutations were analyzed by direct sequencing. RESULTS: In our oncocytic tumor samples, we identified rare TP53 mutations. The series of analyzed cases did not include poorly- or undifferentiated thyroid carcinomas, and none of the TP53 mutated cases had significant mitotic activity or high-grade features. Thus, the presence of disruptive TP53 mutations was completely unexpected. In addition, novel mutations in nuclear-encoded complex I genes were identified. CONCLUSIONS: These findings suggest that nuclear genetic lesions altering the bioenergetics competence of thyroid cells may give rise to an aberrant mitochondria-centered compensatory mechanism and ultimately to the oncocytic phenotype.

Mitochondrial DNA genotyping efficiently reveals clonality of synchronous endometrial and ovarian cancers.

Simultaneous independent primary tumors of the female genital tract occur in 1-2% of gynecological cancer patients, 50-70% of which are synchronous tumors of the endometrium and ovary. Recognition of synchrony upon multiple tumors is crucial for correct prognosis, therapeutic choice, and patient management. Current guidelines for determining synchrony, based on surgical and histopathological findings, are often ambiguous and may require further molecular analyses. However, because of the uniqueness of each tumor and of its intrinsic heterogeneity, these analyses may sometimes be inconclusive. A role for mitochondrial DNA genotyping was previously demonstrated in the diagnosis of synchronous endometrial and ovarian carcinoma. We have analyzed 11 sample pairs of simultaneously revealed endometrial and ovarian cancers and have thereby applied conventional histopathological criteria, current molecular analyses (microsatellite instability, ß-catenin immunohistochemical staining/CTNNB1 mutation screening), and mitochondrial DNA sequencing to distinguish separate independent tumors from metastases, comparing the performance and the informative potential of such methods. We have demonstrated that in ambiguous interpretations where histopathological criteria and canonical molecular methods fail to be conclusive, mitochondrial DNA analysis may act as a needle of balance and allow to formulate a diagnosis in 45.5% of our cases. Additional advantages of mitochondrial DNA genotyping, besides the high level of information we demonstrated here, are the easy implementation and the need for small amounts of starting material. Our results show that mitochondrial DNA genotyping may provide a substantial contribution to indisputably recognize the metastatic nature of simultaneously detected endometrial and ovarian cancers and may change the final staging and clinical management of these patients.

Dysregulation of a Subset of Circulating and Vesicle-Associated miRNA in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive neoplasia, characterized by early metastasis, low diagnostic rates at early stages, resistance to drugs, and poor prognosis. There is an urgent need to better characterize this disease in order to identify efficient diagnostic/prognostic biomarkers. Since microRNAs (miRNAs) contribute to oncogenesis and metastasis formation in PDAC, they are considered potential candidates for fulfilling this task. In this work, the levels of two miRNA subsets (involved in chemoresistance or with oncogenic/tumor suppressing functions) were investigated in a panel of PDAC cell lines and liquid biopsies of a small cohort of patients. We used RT-qPCR and droplet digital PCR (ddPCR) to measure the amounts of cellular- and vesicle-associated, and circulating miRNAs. We found that both PDAC cell lines, also after gemcitabine treatment, and patients showed low amounts of cellular-and vesicle-associated miR-155-5p, compared to controls. Interestingly, we did not find any differences when we analyzed circulating miR-155-5p. Furthermore, vesicle-related miR-27a-3p increased in cancer patients compared to the controls, while circulating let-7a-5p, miR-221-3p, miR-23b-3p and miR-193a-3p presented as dysregulated in patients compared to healthy individuals. Our results highlight the potential clinical significance of these analyzed miRNAs as non-invasive diagnostic molecular tools to characterize PDAC.

A platform independent RNA-Seq protocol for the detection of transcriptome complexity.

BACKGROUND: Recent studies have demonstrated an unexpected complexity of transcription in eukaryotes. The majority of the genome is transcribed and only a little fraction of these transcripts is annotated as protein coding genes and their splice variants. Indeed, most transcripts are the result of antisense, overlapping and non-coding RNA expression. In this frame, one of the key aims of high throughput transcriptome sequencing is the detection of all RNA species present in the cell and the first crucial step for RNA-seq users is represented by the choice of the strategy for cDNA library construction. The protocols developed so far provide the utilization of the entire library for a single sequencing run with a specific platform. RESULTS: We set up a unique protocol to generate and amplify a strand-specific cDNA library representative of all RNA species that may be implemented with all major platforms currently available on the market (Roche 454, Illumina, ABI/SOLiD). Our method is reproducible, fast, easy-to-perform and even allows to start from low input total RNA. Furthermore, we provide a suitable bioinformatics tool for the analysis of the sequences produced following this protocol. CONCLUSION: We tested the efficiency of our strategy, showing that our method is platform-independent, thus allowing the simultaneous analysis of the same sample with different NGS technologies, and providing an accurate quantitative and qualitative portrait of complex whole transcriptomes.

Placing mitochondrial DNA mutations within the progression model of type I endometrial carcinoma.

Mitochondrial DNA (mtDNA) mutations have been described in almost all types of cancer. However, their exact role and timing of occurrence during tumor development and progression are still a matter of debate. A Vogelstein-like model of progression is well established for endometrial carcinoma (EC), however, mtDNA has been scarcely investigated in these tumors despite the fact that mitochondrial biogenesis increase has been shown to be a hallmark of type I EC. Here, we screened a panel of 23 type I EC tissues and matched typical hyperplasia for mutations in mtDNA and in four oncosupressors/oncogenes, namely PTEN, KRAS, CTNNB1 and TP53. Overall, mtDNA mutations were identified in 69% of cases, while mutational events in nuclear genes occurred in 56% of the cases, indicating that mtDNA mutations may precede the genetic instability of these genes canonically involved in progression from hyperplasia to tumor. Protein expression analysis revealed an increase in mitochondrial biogenesis and activation of oxidative stress response mechanisms in tumor tissues, but not in hyperplasia, in correlation with the occurrence of pathogenic mtDNA mutations. Our results point out an involvement of mtDNA mutations in EC progression and explain the increase in mitochondrial biogenesis of type I EC. Last, since mtDNA mutations occur after hyperplasia, their potential role in contributing to genetic instability may be envisioned.

Phenotyping neuroblastoma cells through intelligent scrutiny of stain-free biomarkers in holographic flow cytometry.

To efficiently tackle certain tumor types, finding new biomarkers for rapid and complete phenotyping of cancer cells is highly demanded. This is especially the case for the most common pediatric solid tumor of the sympathetic nervous system, namely, neuroblastoma (NB). Liquid biopsy is in principle a very promising tool for this purpose, but usually enrichment and isolation of circulating tumor cells in such patients remain difficult due to the unavailability of universal NB cell-specific surface markers. Here, we show that rapid screening and phenotyping of NB cells through stain-free biomarkers supported by artificial intelligence is a viable route for liquid biopsy. We demonstrate the concept through a flow cytometry based on label-free holographic quantitative phase-contrast microscopy empowered by machine learning. In detail, we exploit a hierarchical decision scheme where at first level NB cells are classified from monocytes with 97.9% accuracy. Then we demonstrate that different phenotypes are discriminated within NB class. Indeed, for each cell classified as NB its belonging to one of four NB sub-populations (i.e., CHP212, SKNBE2, SHSY5Y, and SKNSH) is evaluated thus achieving accuracy in the range 73.6%-89.1%. The achieved results solve the realistic problem related to the identification circulating tumor cell, i.e., the possibility to recognize and detect tumor cells morphologically similar to blood cells, which is the core issue in liquid biopsy based on stain-free microscopy. The presented approach operates at lab-on-chip scale and emulates real-world scenarios, thus representing a future route for liquid biopsy by exploiting intelligent biomedical imaging.