T cell-based immunotherapies, including immune checkpoint blockade and chimeric antigen receptor (CAR) T cells, have transformed outcomes in several cancers. Yet their effectiveness is routinely undermined by two barriers built by the tumor itself. The first is intrinsic resistance: tumor cells downregulate antigen presentation and display checkpoint molecules such as PD-L1 and CD73 that shut down approaching T cells. The second is the tumor microenvironment itself, which becomes dominated by M2-like tumor-associated macrophages, cells that secrete immunosuppressive cytokines, impair antigen presentation, and actively suppress anti-tumor responses. Most therapeutic strategies address one barrier at a time; the new work demonstrates that a single microRNA can act on both.

The researchers focused on microRNAs, short non-coding RNA molecules of roughly 22 nucleotides that bind complementary sequences in messenger RNAs and dampen protein production. Because each microRNA can regulate dozens of targets at once, they are uniquely positioned to orchestrate broad, coordinated changes in cell behavior, a property the team set out to exploit deliberately. Their central question was whether miR-155-5p, and a related candidate called miR-3535, could drive functional reprogramming of both tumor cells and macrophages in a concerted fashion.

Experimentally, the approach was straightforward but comprehensive. Human tumor cell lines drawn from several different cancer entities were transfected with synthetic miR-155-5p or miR-3535, and the resulting changes in immune checkpoint molecule expression and cell proliferation were measured at both the transcript and protein level. In parallel, M2-polarized macrophages generated from peripheral blood mononuclear cells of healthy donors received the same microRNA treatment. The team then profiled cytokine secretion by enzyme-linked immunosorbent assay and carried out transcriptomic analysis, combining RNA sequencing with microarray-based gene expression profiling, to map the macrophage polarization states and immune-regulatory pathways altered by treatment. Transcription factor activity and gene set enrichment analyses were used to identify the regulatory circuits at work.

The results in tumor cells were striking. Both microRNAs reduced expression of CD73, encoded by the NT5E gene, an ectoenzyme that degrades extracellular ATP into immunosuppressive adenosine and is widely regarded as a driver of tumor immune escape. miR-155-5p went further, also suppressing PD-L1 (CD274), the ligand targeted by some of the most widely used checkpoint inhibitor drugs. Knocking down both molecules in a single step effectively removes two of the brakes tumors place on T cells, one that blocks T cell activation through the PD-1 axis and one that poisons the metabolic environment around the tumor.

The macrophage findings were equally significant. When M2-like macrophages, the immunosuppressive, wound-healing subtype that accumulates in tumors, were transfected with either microRNA, they shifted toward a pro-inflammatory M1-like phenotype. This conversion was measurable functionally: treated macrophages secreted markedly more TNFα, a cytokine with direct anti-tumor activity, and CXCL10, a chemokine that recruits activated T cells into tissues. Gene expression analysis confirmed the induction of M1-associated genes across the board.

The transcriptomic data revealed the mechanism in finer detail. MicroRNA treatment activated inflammatory signaling pathways driven by STAT1, a signal transducer and activator of transcription, and by interferon regulatory factors, the downstream effectors of interferon signaling that define the classical inflammatory macrophage state. At the same time, the activity of ZNF703, a zinc finger transcription factor that the study identifies as a transcriptional hub associated with M2 macrophage infiltration and poor clinical prognosis, was reduced. In other words, the microRNAs did not merely nudge macrophages; they flipped the regulatory logic of the cell, amplifying the inflammatory program while simultaneously quieting a master regulator of the tumor-permissive state.

A further observation points to a possible bonus effect on the anti-tumor immune response itself. Both microRNAs increased expression of TAP1, the transporter associated with antigen processing 1, a critical component of the machinery that loads peptide fragments onto MHC class I molecules for display to cytotoxic T cells. Enhanced TAP1 expression suggests improved antigen-processing capacity, potentially making tumor cells and antigen-presenting cells more visible to the immune system. This is particularly relevant because loss of antigen presentation is a well-documented route by which tumors escape both natural immune surveillance and T cell-based therapies.

Beyond their immunological effects, both microRNAs exerted direct anti-proliferative effects across tumor cell lines from multiple entities. That the same molecule slows tumor growth while simultaneously reversing checkpoint expression and repolarizing macrophages is what distinguishes this work from more narrowly targeted approaches. The findings link tumor cell plasticity to neutralization of the immunosuppressive tumor environment within a single regulatory mechanism, rather than treating these as separate problems requiring separate drugs.

The broader implications for cancer immunotherapy are considerable. Current strategies to overcome immune resistance typically involve combining checkpoint inhibitors with each other or with chemotherapy, radiation, or macrophage-targeting agents, an approach that multiplies toxicity and cost. A microRNA-based strategy that acts on several fronts at once could, in principle, simplify this combinatorial challenge. The authors note that the findings support further investigation of microRNA-based strategies in cancer immunotherapy, and the field has already developed delivery tools, including lipid nanoparticles, that could in theory carry synthetic microRNAs to tumors and tumor-associated immune cells in vivo.

Caution is warranted, as always in preclinical work. The experiments were conducted in cell lines and in donor-derived macrophages, not in patients, and the challenge of delivering a microRNA selectively to the right cells in a living tumor remains formidable. miR-155 in particular is a pleiotropic molecule with roles in inflammation and immunity that cut both ways; systemic elevation could carry inflammatory risks, and past clinical experience with nucleic acid therapeutics has taught the field to be skeptical of simple delivery assumptions. The question of dose, timing, and tissue specificity will need to be answered in animal models and, eventually, carefully designed clinical studies.

Even so, the conceptual contribution is substantial. The study demonstrates that microRNAs are capable of coordinating anti-tumor effects across different cell types, a property that individual protein-targeting drugs rarely possess. If the coordinated reprogramming seen in vitro can be reproduced in vivo, miR-155-5p and miR-3535 would represent a template for a new class of immunotherapy, one that does not simply block a single checkpoint or deplete a single cell population, but rewires the conversation between tumor and immune system at multiple points simultaneously. At a time when the majority of patients still do not benefit from existing immunotherapies, strategies that address tumor-intrinsic resistance and microenvironmental suppression in one stroke are exactly the kind of innovation the field has been searching for.

The core insight of the review is that focused ultrasound, or FUS, does far more than heat tissue. When an acoustic beam is focused to a small target volume, it deposits energy through three broadly distinct mechanisms: mechanical effects driven by acoustic radiation forces, cavitation-driven effects arising from the dynamics of microscopic gas bubbles, and thermal effects from the absorption of ultrasound energy. Cavitation is particularly dramatic. When pre-existing or newly formed microbubbles oscillate and then implode under acoustic pressure, they generate localized regions of extreme pressure and temperature, producing shockwaves and microjets that can stretch the cell membrane into transient, tiny pores—a phenomenon called sonoporation that allows molecules and ions to pass through without permanently damaging the cell. In parallel, acoustic radiation forces transfer momentum to tissue during sound propagation, displacing and deforming cell membranes at the focal point, while acoustic streaming induces steady shear stresses that further perturb cellular and subcellular structures.

These physical perturbations are not simply destructive; they are informative. The Rice team emphasizes that cells interpret FUS-induced mechanical forces through mechanotransduction—the conversion of mechanical stimuli into biochemical signals. Matrix-anchored cells detect these disturbances through the integrin-adhesion plaque complex, transmitting them along actin stress fibers, while suspended cells experience shear force directly at the plasma membrane. Forces propagating through the cytoskeleton can even reach the nucleus via the linker of nucleoskeleton and cytoskeleton complex, influencing chromatin organization and gene expression. But the most striking mechanistic story involves mechanosensitive ion channels. PIEZO1 has been repeatedly identified as a primary mechano-gated channel responsive to acoustic radiation force-driven membrane tension: low-intensity FUS rapidly activates PIEZO1-dependent calcium influx in osteoblastic precursor cells, promoting ERK signaling and cytoskeletal remodeling, while in prostate cancer models nonthermal ultrasound pulses induce PIEZO1-mediated calcium entry that causes mitochondrial depolarization and caspase-3 activation, sensitizing tumors to TRAIL-mediated apoptosis. The TRPV4 channel, meanwhile, has emerged as a key sonosensor at the blood-brain barrier, where cavitation and radiation force-induced membrane strain gates TRPV4-dependent calcium influx, engaging a Ca²⁺/PKC-δ cascade that drives reversible tight-junction opening. Two-pore domain potassium channels such as TREK-1 and TRAAK add another dimension, converting FUS-induced membrane tension into hyperpolarizing leak currents that dampen neuronal excitability—in one remarkable study, transcranial low-intensity FUS targeting TRAAK-overexpressing brain neurons suppressed sympathetic drive and prevented malignant arrhythmias after myocardial infarction.

The therapeutic implications of this channel-level control are profound. Calcium signaling is a master regulator of cell fate, and FUS can push it in either direction depending on acoustic parameters. Low-intensity pulsed ultrasound enhances tissue regeneration and migration, whereas elevated mechanical forces trigger apoptosis through extensive DNA damage or altered mitochondrial permeability. In hepatocellular carcinoma models, FUS stimulation suppressed tumor proliferation by more than 70 percent in H22-HCC cells and more than 83 percent in Hepa1-6-HCC cells, along with significantly prolonged survival. In immunotherapy contexts, high-intensity ultrasound triggered the calcium-dependent NFAT pathway in T cells, producing stronger immune responses and memory that effectively inhibited tumor recurrence and metastasis. The review also highlights FUS’s capacity to transiently and locally open the blood-brain barrier—a critical translational goal, since passive diffusion across the barrier typically favors only small lipophilic molecules under roughly 400 to 500 Daltons, yet nearly 98 percent of approved small-molecule drugs exceed this threshold. Preclinical and early clinical studies of FUS-mediated BBB opening demonstrate spatially defined, reversible increases in regional permeability that allow therapeutic agents and biologics to access previously restricted brain regions.

The second pillar of the framework concerns nanoparticles as active partners rather than passive cargo holders. Compared with microbubbles—which are 1 to 8 micrometers in diameter, confined largely to vascular compartments, limited in drug-loading capacity, and short-lived in circulation—nanoparticles in the 20 to 200 nanometer range offer tunable size, broad surface functionalization, prolonged systemic circulation, and the ability to access extravascular and interstitial spaces. Crucially, nanoparticles can be engineered as transducers that convert acoustic cues into on-demand structural reconfiguration or bond cleavage. The review catalogs three classes of FUS-triggered chemical bond scission. Surface-anchoring bonds can be severed to shed protective shells: silica core-shell nanoparticles bearing a PEG brush attached via force-labile azo bonds remain stable during circulation until FUS-induced mechanical perturbation triggers PEG detachment, activating free radical generation and cytotoxicity. Prodrug-linker bonds embed sono-labile chemistry at the drug-carrier junction: singlet oxygen generated by therapeutic ultrasound can cleave a urea linkage between carboxyferrocene and methylene blue, switching an inert nanodrug into a Fenton-active ferroptosis inducer at the tumor site. Backbone and crosslink bonds determine whether ultrasound destabilizes the entire carrier framework, as in diselenide-crosslinked microgels that degrade into water-soluble chains under low-frequency ultrasound, or thermosensitive hydrogels that disintegrate under mild FUS hyperthermia to release ultrasmall 1-to-5-nanometer doxorubicin-loaded secondary nanoparticles deep into tumor microvasculature.

Not all FUS-nanoparticle interactions require covalent bond rupture, however. The review details reversible physical mechanisms in which ultrasound controls membrane properties, aggregation states, or spatial distribution without permanent chemical modification. Thermosensitive liposomal bilayers tuned with DPPC/DSPC/MSPC compositions remain stable at 37 degrees Celsius but generate transient membrane defects under mild FUS hyperthermia, accelerating release of encapsulated carboplatin and membrane-associated SN-38. In a triple-negative breast cancer model, FUS-triggered doxorubicin liposomes increased vascular permeability, promoted immunogenic cell death, and reprogrammed a suppressive tumor microenvironment into an immune-responsive one that enhanced checkpoint blockade efficacy. Piezoelectric barium titanate nanoparticles activated by FUS generate reactive oxygen species or trigger nitric oxide release, altering stromal components such as collagen and fibronectin—demonstrating that nanoparticles can actively reshape the tumor microenvironment in concert with acoustic stimulation.

The third and perhaps most forward-looking pillar of the review is its argument for advanced in vitro testing platforms. The authors note that the National Institutes of Health has recently shifted research priorities toward human-based technologies, establishing the Office of Research Innovation, Validation, and Application to reduce reliance on animal models, which frequently fail to translate—many candidate therapies fail in phase I and II clinical trials despite promising rodent results, owing to fundamental interspecies differences in metabolism, molecular interactions, and disease progression. Conventional two-dimensional cell culture fares no better: flat, rigid substrates cannot capture the three-dimensional multicellular architecture of tumors, the mechanical cues of extracellular matrix stiffness, or the cell-cell interactions—including bidirectional mitochondrial transfer between cancer and immune cells—that regulate therapeutic response. Intermediate systems such as Transwell chambers, 3D hydrogel cultures, and tumor organoids each address parts of this gap, but they remain limited in their ability to support controlled perfusion and spatiotemporal regulation.

Cancer-on-a-chip platforms close this remaining gap. These microfluidic systems integrate self-assembled vascular networks, defined extracellular matrix structures, and regulated flow within optically accessible formats, enabling real-time, quantitative analysis of nanoparticle penetration, distribution, and release under physiologically controlled conditions. The review describes how vascularized chip models—including glioblastoma-on-a-chip systems—allow assessment of nanodrug formulations designed to preserve vascular integrity during FUS exposure, while stiffness-tunable hydrogel microfluidic systems reveal how matrix mechanics regulate cancer cell migration and invasion. When FUS is incorporated directly into these chips, researchers can resolve in real time how acoustic stimulation, nanoparticle activation, and tumor-vascular-immune interactions couple together—effects that static culture systems average away and that xenograft models obscure. One cited study integrated FUS with microbubble oscillation in an organ-on-chip model to disrupt the extracellular matrix and enhance interstitial drug transport, while other work showed FUS activating microglia, hinting at immune modulation possibilities in brain tumors.

The authors are candid about the challenges that remain. The effective and safe ultrasound dose range for combined FUS-nanoparticle therapy, as well as repeated dosing strategies, is still unclear, and complex multicomponent formulations need standardization for large-scale production, quality control, and regulatory approval. Yet the trajectory is clear: next-generation cancer-on-a-chip platforms that reconstruct vascular perfusion, matrix mechanics, immune infiltration, and a tunable field for FUS stimulation—ideally built from heterogeneous patient samples—could serve as a translational bridge from nanomedicine design to clinical implementation, ultimately enabling personalized assessment of FUS-responsive therapies. If that bridge is crossed, the humble sound wave, working in concert with engineered nanoparticles, could become one of the most versatile tools in oncology: a knife-less surgeon, a courier for drugs, and a reprogrammer of the tumor microenvironment, all in one focused beam.

The study presents a compelling case for the triple remodeling of the tumor microenvironment, a strategy that focuses on enhancing drug delivery while simultaneously disrupting the protective barriers that tumors utilize to evade immune surveillance. By employing lipid nanoparticles that are specifically designed to target folate receptors—a hallmark of many cancer cells—the delivery system significantly increases the uptake of therapeutic agents within malignant tissues. This mechanism hinges upon the ability of these nanoparticles to navigate the complex cellular landscape of tumors, ensuring that they release their cargo where it is needed most.

Central to this research is the dual-targeting of both VEGF (vascular endothelial growth factor) and PD-L1 (programmed death-ligand 1). VEGF is known for its role in promoting angiogenesis, thereby supporting tumor growth and survival. By silencing VEGF via siRNA, the researchers can potentially debilitate the tumor’s blood supply, depriving it of necessary nutrients and oxygen. On the other hand, the targeted modulation of PD-L1 serves to enhance the efficacy of T-cell responses against cancer cells, thereby facilitating a more vigorous immune attack.

Hyaluronidase has been strategically incorporated into this experimental framework, serving as an enabler for enhanced drug penetration and distribution within dense tumor stroma. By breaking down hyaluronic acid—a major component of the extracellular matrix—hyaluronidase effectively reduces barriers to diffusion, allowing the therapeutic nanoparticles to infiltrate deeper into tumor tissues where conventional therapies struggle to reach. This enzymatic remodeling represents a significant shift in the approach to chemotherapeutic and immunotherapeutic delivery.

The implications of this study extend beyond the immediate effects on tumor behavior. By utilizing this targeted combination therapy, researchers envision an environment in which tumors are not only subject to direct cytotoxic effects but are also reconditioned to become more “immunogenic”. Through this process, cancer cells may be reprogrammed to express more immunogenic markers, thereby attracting immune responses that have previously been thwarted by tumor evasion tactics.

Moreover, the findings contribute to a growing body of literature emphasizing the importance of the tumor microenvironment in dictating therapeutic outcomes. The study positions the tumor microenvironment not merely as a passive background but as an active player in cancer biology—one that can be strategically manipulated to favor therapeutic efficacy. This perspective warrants a paradigm shift in the design of future cancer treatment protocols, accommodating the nuanced interactions between cancer cells and their surrounding environment.

The research methodology employed in this study is equally noteworthy. The authors utilized rigorous in vitro and in vivo models to validate their hypotheses, employing advanced imaging techniques to track nanoparticle distribution and silencing efficiency across different tumor types. These methodologies not only affirm the robustness of the results but also pave the way for future investigations into various theranostic applications—therapeutic regimens that also deliver diagnostic capabilities.

Furthermore, it’s essential to contextualize these findings within the broader landscape of cancer immunotherapy. Current immune checkpoint inhibitors, while promising, have shown varied responses among patients. The integration of a multi-faceted approach, as demonstrated in this study, could enhance the predictability of patient responses, leading to more personalized and effective treatment plans. This personalized medicine approach aligns closely with contemporary trends in oncology, catering to the unique biological dynamics exhibited by individual tumors.

As the research community continues to navigate the complexities of cancer treatment, the work by Li et al. stands out as a hallmark of innovative thinking. Their insights lay a compelling groundwork for future clinical trials aimed at translating these findings into standard medical practice. The potential to combine targeted therapy with immunology-enhancing strategies could revolutionize how patients respond to cancer treatment, making once-difficult-to-treat tumors more manageable.

In summary, the research highlights the significant promise of combining hyaluronidase-assisted strategies with advanced lipid nanoparticle technology to achieve a new standard in tumor immunotherapy. The intricate interplay between treatment modalities exemplified in this study represents a promising avenue for enhanced therapeutic efficacy in oncology. As researchers build upon this foundation, a future where cancer treatments are more effective and personalized is not just a possibility—it’s becoming a reality.

This pioneering study has set the stage for further exploration and validation within clinical settings, and with the potential to improve patient outcomes significantly, it underscores the importance of continued vigilance and innovation within the realm of cancer therapeutics. Exciting advancements and nuanced understandings of cancer biology are on the horizon, and the efforts put forth by Li et al. may very well lead the charge toward transformative solutions in the fight against cancer.

The global scientific community eagerly anticipates the developments that will arise from this foundational work, as the translational impact of their findings could very well reshape our understanding and approach to cancer treatment in years to come.

In conclusion, the integration of innovative methodologies and dual-targeting strategies not only enriches the current discourse on cancer immunotherapy but also positions the research efforts of Li et al. at the forefront of the ongoing battle against cancer, offering renewed hope and potential pathways for patients battling this pervasive disease.

Subject of Research: Triple remodeling of tumor microenvironment for cancer immunotherapy

Article Title: Triple-remodeling of tumor microenvironment through hyaluronidase-assisted folate-targeted lipid nanoparticle-mediated siVEGF/siPD-L1 for enhanced tumor immunotherapy

Article References:

Li, X., Xue, H., Liu, Q. et al. Triple-remodeling of tumor microenvironment through hyaluronidase-assisted folate-targeted lipid nanoparticle-mediated siVEGF/siPD-L1 for enhanced tumor immunotherapy.
J Transl Med (2026). https://doi.org/10.1186/s12967-026-07697-y

Image Credits: AI Generated

DOI: 10.1186/s12967-026-07697-y

Keywords: immunotherapy, lipid nanoparticles, tumor microenvironment, VEGF, PD-L1, hyaluronidase, siRNA, cancer treatment

The concept behind this study stems from the understanding that solid tumors often develop mechanisms to circumvent detection and elimination by the immune system. In a typical healthy individual, T cells, a critical component of the immune response, identify and attack cancer cells by releasing interferon-gamma, a potent cytokine. This cytokine has the ability to inhibit tumor growth and activate various immune pathways. However, its effectiveness has been poorly translated into many cancer treatments, owing largely to the ability of certain tumors to develop resistance. The researchers at St. Jude have discovered how VDAC2 helps these tumors resist the effects of interferon-gamma.

By conducting CRISPR-Cas9 screenings, the team was able to pinpoint the genes most implicated in tumor immunoresistance. Their subsequent findings showcased that the absence of VDAC2 significantly increased cancer cell susceptibility to interferon-gamma and led to a marked increase in tumor cell death. This absence not only promoted the direct killing of cancer cells but also incited an inflammatory response that rendered tumors more amenable to treatment with immunotherapeutic agents. Such findings are particularly pivotal, as they provide a pathway for the development of new drugs targeting proteins like VDAC2 that protect tumor cells by preventing their demise and blocking inflammatory signals.

In essence, the researchers have likened VDAC2 to a signal jammer for mobile phones, obstructing essential communications between tumor cells and the immune system. When VDAC2 is present, it prevents tumor cells from receiving the critical signals sent by T cells, thereby stunting the immune response. The compelling aspect of this study is that by eliminating VDAC2, the cancer cells are better positioned to detect and respond to these anticancer signals, which in turn leads to the activation of pathways that promote cell death.

Another significant finding from this study reveals the undeservedly prominent role of mitochondria in immune responses against tumors. Once viewed primarily as energy-producing organelles, mitochondria exhibit far-reaching implications on cell signaling, particularly in terms of immune communication. When VDAC2 is knocked out in cancer cells, these cells can release mitochondrial DNA that activates specific immune sensors, ultimately stimulating the production of type I interferons. This cascade results in a heightened immune response, effectively providing the cancer cells with the means to communicate with the body’s adaptive immune system, thus amplifying the anticancer effects.

The relationship between cytokines and mitochondrial activity highlights a complex interplay in regulating immune responses within tumors. Researchers observed that when VDAC2 was absent, there was an unexpected enhancement of innate immune signaling that stemmed from T-cell activities. This reverse mechanism invites further exploration into the development of novel therapies aimed at modifying mitochondrial functions in tumors to aid immunotherapy.

While VDAC2 presents an attractive target for therapeutic intervention, it is crucial to note that no specific drugs currently exist for its inhibition. However, this study lays the groundwork for future research efforts directed toward developing such inhibitors. The increasing understanding of how VDAC2 operates within the tumor microenvironment and its implications for immunotherapy opens new avenues for treatment strategies.

As a culmination of their findings, St. Jude researchers have put forth a new framework for identifying drug targets within tumors. By focusing on signaling pathways that serve dual purposes in protecting tumor cells, the hope is to enhance the efficacy of immunotherapies and, in turn, improve patient outcomes. The prevailing challenge remains to translate these laboratory findings into viable clinical interventions that can be routinely applied to treat resistant cancers.

Moreover, the ongoing exploration of mitochondrial biology in the context of cancer treatment could unlock revolutionary approaches for overcoming the barriers that have thwarted traditional therapies for decades. Understanding how to harness this knowledge could prove pivotal in the quest to find effective treatments that can one day enable all patients to benefit from the miraculous potential of immunotherapy.

The research team, led by corresponding author Hongbo Chi, emphasizes the urgency for stakeholders in the scientific community to take note of these discoveries. As novel therapeutic strategies emerge, it will be essential to collaborate across various specialties in cancer research and immunology to ensure that we capitalize on the insights gained from these preliminary studies. The future of cancer immunotherapy could very well hinge on the ability to exploit the vulnerabilities uncovered in this research focusing on VDAC2 and its critical role in tumor resilience.

Without a doubt, this study is a reminder that breakthroughs in cancer research often arise from the most unexpected findings, encouraging researchers to continually question established paradigms in cancer treatment. With the findings from St. Jude Children’s Research Hospital, the message to the scientific community is clear: there are still unknowns in the complex interplay between cancer cells and the immune system, and uncovering these intricacies can pave the way towards more effective strategies for eradicating cancer.

Subject of Research: VDAC2 and its role in tumor immunology
Article Title: VDAC2 loss unleashes tumor destruction and inflammation for cancer therapy
News Publication Date: 19-Mar-2025
Web References: St. Jude Children’s Research Hospital
References: Nature Journal
Image Credits: Courtesy of St. Jude Children’s Research Hospital

Keywords: Cancer immunotherapy, Gene targeting, Molecular targets, Cell death pathways, Cancer cells, Cellular proteins, Mitochondrial proteins, Tumor cells, Cancer research, Mitochondrial DNA