A novel multi-stage enrichment workflow and comprehensive characterization for HEK293F-derived extracellular vesicles.

Extracellular vesicles (EVs) are emerging as a promising drug delivery vehicle as they are biocompatible and capable of targeted delivery. However, clinical translation of EVs remains challenging due to the lack of standardized and scalable manufacturing protocols to consistently isolate small EVs (sEVs) with both high yield and high purity. The heterogenous nature of sEVs leading to unknown composition of biocargos causes further pushback due to safety concerns. In order to address these issues, we developed a robust quality-controlled multi-stage process to produce and isolate sEVs from human embryonic kidney HEK293F cells. We then compared different 2-step and 3-step workflows for eliminating protein impurities and cell-free nucleic acids to meet acceptable limits of regulatory authorities. Our results showed that sEV production was maximized when HEK293F cells were grown at high-density stationary phase in semi-continuous culture. The novel 3-step workflow combining tangential flow filtration, sucrose-cushion ultracentrifugation and bind-elute size-exclusion chromatography outperformed other methods in sEV purity while still preserved high yield and particle integrity. The purified HEK293F-derived sEVs were thoroughly characterized for identity including sub-population analysis, content profiling including proteomics and miRNA sequencing, and demonstrated excellent preclinical safety profile in both in-vitro and in-vivo testing. Our rigorous enrichment workflow and comprehensive characterization will help advance the development of EVs, particularly HEK293F-derived sEVs, to be safe and reliable drug carriers for therapeutic applications.

Analytical validation and clinical utilization of K-4CARE™: a comprehensive genomic profiling assay with personalized MRD detection.

Background: Biomarker testing has gradually become standard of care in precision oncology to help physicians select optimal treatment for patients. Compared to single-gene or small gene panel testing, comprehensive genomic profiling (CGP) has emerged as a more time- and tissue-efficient method. This study demonstrated in-depth analytical validation of K-4CARE, a CGP assay that integrates circulating tumor DNA (ctDNA) tracking for residual cancer surveillance. Methods: The assay utilized a panel of 473 cancer-relevant genes with a total length of 1.7 Mb. Reference standards were used to evaluate limit of detection (LOD), concordance, sensitivity, specificity and precision of the assay to detect single nucleotide variants (SNVs), small insertion/deletions (Indels), gene amplification and fusion, microsatellite instability (MSI) and tumor mutational burden (TMB). The assay was then benchmarked against orthogonal methods using 155 clinical samples from 10 cancer types. In selected cancers, top tumor-derived somatic mutations, as ranked by our proprietary algorithm, were used to detect ctDNA in the plasma. Results: For detection of somatic SNVs and Indels, gene fusion and amplification, the assay had sensitivity of >99%, 94% and >99% respectively, and specificity of >99%. Detection of germline variants also achieved sensitivity and specificity of >99%. For TMB measurement, the correlation coefficient between whole-exome sequencing and our targeted panel was 97%. MSI analysis when benchmarked against polymerase chain reaction method showed sensitivity of 94% and specificity of >99%. The concordance between our assay and the TruSight Oncology 500 assay for detection of somatic variants, TMB and MSI measurement was 100%, 89%, and 98% respectively. When CGP-informed mutations were used to personalize ctDNA tracking, the detection rate of ctDNA in liquid biopsy was 79%, and clinical utility in cancer surveillance was demonstrated in 2 case studies. Conclusion: K-4CARE™ assay provides comprehensive and reliable genomic information that fulfills all guideline-based biomarker testing for both targeted therapy and immunotherapy. Integration of ctDNA tracking helps clinicians to further monitor treatment response and ultimately provide well-rounded care to cancer patients.

Skin rejuvenation and photoaging protection using adipose-derived stem cell extracellular vesicles loaded with exogenous cargos.

BACKGROUND: Small extracellular vesicles from adipose-derived stem cells (ASC-sEVs) have gained remarkable attention for their regenerative and protective properties against skin aging. However, the use of ASC-sEVs to further encapsulate certain natural anti-aging compounds for synergistic effects has not been actively explored. For large-scale production in skincare industry, it is also crucial to standardize cost-effective methods to produce highly pure ASC-sEVs. METHODS: Human ASCs were expanded in serum-free media with different compositions to first optimize the sEV production. ASC-sEVs from different batches were then purified using tangential flow filtration and sucrose cushion ultracentrifugation, followed by extensive characterization for identity and content profiling including proteomics, lipidomics and miRNA sequencing. ASC-sEVs were further loaded with nicotinamide riboside (NR) and resveratrol by sonication-incubation method. The therapeutic effect of ASC-sEVs and loaded ASC-sEVs was tested on human keratinocyte cell line HaCaT exposed to UVB by measuring reactive oxygen species (ROS). The loaded ASC-sEVs were later applied on the hand skin of three volunteers once a day for 8 weeks and skin analysis was performed every 2 weeks. RESULTS: Our standardized workflow produced ASC-sEVs with high yield, high purity and with stable characteristics and consistent biocargo among different batches. The most abundant subpopulations in ASC-sEVs were CD63+ (∼30%) and CD81+ -CD63+ (∼35%). Purified ASC-sEVs could be loaded with NR and resveratrol at the optimized loading efficiency of ∼20%. In UVB-exposed HaCaT cells, loaded ASC-sEVs could reduce ROS by 38.3%, higher than the sEVs (13.3%) or compounds (18.5%) individually. In human trial, application of loaded ASC-sEVs after 8 weeks substantially improved skin texture, increased skin hydration and elasticity by 104% and reduced mean pore volume by 51%. CONCLUSIONS: This study demonstrated a robust protocol to produce ASC-sEVs and exogenously load them with natural compounds. The loaded ASC-sEVs exhibited synergistic effects of both sEVs and anti-aging compounds in photoaging protection and skin rejuvenation.

Dual-targeting exosomes for improved drug delivery in breast cancer.

Aims: The authors investigated whether displaying more than one homing peptide enhanced the tumor-targeting efficiency of exosomes. Materials & methods: Exosomes from human embryonic kidney cells (HEK293F) were engineered to display either mono- or dual-tumor-penetrating peptides, iRGD and tLyp1. Exosomes were purified via tangential flow filtration followed by ultracentrifugation. Results: When loaded with doxorubicin (Dox), the dual iRGD-tLyp1 exosomes strongly enhanced Dox uptake in both MCF-7 and MDA-MB-231 breast cancer cell lines, superior to single iRGD or tLyp1 exosomes. The dual iRGD-tLyp1 exosomal Dox was also the most potent, with IC50/GI50 values being 3.7-17.0-times lower than those of free Dox and other exosomal Dox. Conclusion: Selecting appropriate combinatorial homing peptides could be an approach for future precision nanomedicine.

Genetic landscape and personalized tracking of tumor mutations in Vietnamese women with breast cancer.

Breast cancer is the leading cause of cancer death in Vietnamese women, but its mutational landscape and actionable alterations for targeted therapies remain unknown. After treatment, a sensitive biomarker to complement conventional imaging to monitor patients is also lacking. In this prospective multi-center study, 134 early-stage breast cancer patients eligible for curative-intent surgery were recruited. Genomic DNA from tumor tissues and paired white blood cells were sequenced to profile all tumor-derived mutations in 95 cancer-associated genes. Our bioinformatic algorithm was then utilized to identify top mutations for individual patients. Serial plasma samples were collected before surgery and at scheduled visits after surgery. Personalized assay tracking the selected mutations were performed to detect circulating tumor DNA (ctDNA) in the plasma. We found that the mutational landscape of the Vietnamese was largely similar to other Asian cohorts, showing higher TP53 mutation frequency than in Caucasians. Alterations in PIK3CA and PI3K signaling were dominant, particularly in our triple-negative subgroup. Using top-ranked mutations, we detected ctDNA in pre-operative plasma in 24.6-43.5% of the hormone-receptor-positive groups and 76.9-80.8% of the hormone-receptor-negative groups. The detection rate was associated with breast cancer subtypes and clinicopathological features that increased the risk of relapse. Interim analysis after a 15-month follow-up revealed post-operative detection of ctDNA in all three patients that had recurrence, with a lead time of 7-13 months ahead of clinical diagnosis. Our personalized assay is streamlined and affordable with promising clinical utility in residual cancer surveillance. We also generated the first somatic variant dataset for Vietnamese breast cancer women that could lay the foundation for precision cancer medicine in Vietnam.

Tumor genomic profiling and personalized tracking of circulating tumor DNA in Vietnamese colorectal cancer patients.

Background: Colorectal cancer (CRC) is the fifth most common cancer with rising prevalence in Vietnam. However, there is no data about the mutational landscape and actionable alterations in the Vietnamese patients. During post-operative surveillance, clinical tools are limited to stratify risk of recurrence and detect residual disease. Method: In this prospective multi-center study, 103 CRC patients eligible for curative-intent surgery were recruited. Genomic DNA from tumor tissue and paired white blood cells were sequenced to profile all tumor-derived somatic mutations in 95 cancer-associated genes. Our bioinformatic algorithm identified top mutations unique for individual patient, which were then used to monitor the presence of circulating tumor DNA (ctDNA) in serial plasma samples. Results: The top mutated genes in our cohort were APC, TP53 and KRAS. 41.7% of the patients harbored KRAS and NRAS mutations predictive of resistance to Cetuximab and Panitumumab respectively; 41.7% had mutations targeted by either approved or experimental drugs. Using a personalized subset of top ranked mutations, we detected ctDNA in 90.5% of the pre-operative plasma samples, whereas carcinoembryonic antigen (CEA) was elevated in only 41.3% of them. Interim analysis after 16-month follow-up revealed post-operative detection of ctDNA in two patients that had recurrence, with the lead time of 4-10.5 months ahead of clinical diagnosis. CEA failed to predict recurrence in both cases. Conclusion: Our assay showed promising dual clinical utilities in residual cancer surveillance and actionable mutation profiling for targeted therapies in CRC patients. This could lay foundation to empower precision cancer medicine in Vietnam and other developing countries.

microRNA Expression Levels Change in Neonatal Patients During and After Exposure to Cardiopulmonary Bypass.

Background The systemic inflammation that occurs after exposure to cardiopulmonary bypass (CPB), which is especially severe in neonatal patients, is associated with poorer outcomes and is not well understood. In order to gain deeper insight into how exposure to bypass activates inflammatory responses in circulating leukocytes, we studied changes in microRNA (miRNA) expression during and after exposure to bypass. miRNAs are small noncoding RNAs that have important roles in modulating protein levels and function of cells. Methods and Results We performed miRNA-sequencing on leukocytes isolated from neonatal patients with CPB (n=5) at 7 time points during the process of CPB, including before the initiation of bypass, during bypass, and at 3 time points during the first 24 hours after weaning from bypass. We identified significant differentially expressed miRNAs using generalized linear regression models, and miRNAs were defined as statistically significant using a false discovery rate-adjusted P<0.05. We identified gene targets of these miRNAs using the TargetScan database and identified significantly enriched biological pathways for these gene targets. We identified 54 miRNAs with differential expression during and after CPB. These miRNAs clustered into 3 groups, including miRNAs that were increased during and after CPB (3 miRNAs), miRNAs that decreased during and after CPB (10 miRNAs), and miRNAs that decreased during CPB but then increased 8 to 24 hours after CPB. A total of 38.9% of the target genes of these miRNAs were significantly differentially expressed in our previous study. miRNAs with altered expression levels are predicted to significantly modulate pathways related to inflammation and signal transduction. Conclusions The unbiased profiling of the miRNA changes that occur in the circulating leukocytes of patients with bypass provides deeper insight into the mechanisms that underpin the systemic inflammatory response that occurs in patients after exposure to CPB. These data will help the development of novel treatments and biomarkers for bypass-associated inflammation.

Functional metabolite reserves and lipid homeostasis revealed by the MA-10 Leydig cell metabolome.

In Leydig cells, intrinsic factors that determine cellular steroidogenic efficiency is of functional interest to decipher and monitor pathophysiology in many contexts. Nevertheless, beyond basic regulation of cholesterol storage and mobilization, systems biology interpretation of the metabolite networks in steroidogenic function is deficient. To reconstruct and describe the different molecular systems regulating steroidogenesis, we profiled the metabolites in resting MA-10 Leydig cells. Our results identified 283-annotated components (82 neutral lipids, 154 membrane lipids, and 47 other metabolites). Neutral lipids were represented by an abundance of triacyglycerols (97.1%), and low levels of cholesterol esters (2.0%). Membrane lipids were represented by an abundance of glycerophospholipids (77.8%), followed by sphingolipids (22.2%). Acylcarnitines, nucleosides, amino acids and their derivatives were the other metabolite classes identified. Among nonlipid metabolites, we recognized substantial reserves of aspartic acid, choline, creatine, betaine, glutamine, homoserine, isoleucine, and pantothenic acid none of which have been previously considered as a requirement in steroidogenic function. Individually limiting use of betaine, choline, or pantothenic acid, during luteinizing hormone-induced steroidogenesis in MA-10 cells resulted in substantial decreases to acute steroidogenic capacity, explained by intermediary metabolite imbalances affecting homeostasis. As such, our dataset represents the current level of baseline characterization and unravels the functional resting state of steroidogenic MA-10 Leydig cells. In identifying metabolite stockpiles and causal mechanisms, these results serve to further comprehend the cellular setup and regulation of steroid biosynthesis.

Pathogenic Variant Profile of Hereditary Cancer Syndromes in a Vietnamese Cohort.

BACKGROUND: Hereditary cancer syndromes (HCS) are responsible for 5-10% of cancer cases. Genetic testing to identify pathogenic variants associated with cancer predisposition has not been routinely available in Vietnam. Consequently, the prevalence and genetic landscape of HCS remain unknown. METHODS: 1165 Vietnamese individuals enrolled in genetic testing at our laboratory in 2020. We performed analysis of germline mutations in 17 high- and moderate- penetrance genes associated with HCS by next generation sequencing. RESULTS: A total of 41 pathogenic variants in 11 genes were detected in 3.2% individuals. The carrier frequency was 4.2% in people with family or personal history of cancer and 2.6% in those without history. The percentage of mutation carriers for hereditary colorectal cancer syndromes was 1.3% and for hereditary breast and ovarian cancer syndrome was 1.6%. BRCA1 and BRCA2 mutations were the most prevalent with the positive rate of 1.3% in the general cohort and 5.1% in breast or ovarian cancer patients. Most of BRCA1 mutations located at the BRCA C-terminus domains and the top recurrent mutation was NM_007294.3:c.5251C>T (p.Arg1751Ter). One novel variant NM_000038.6(APC):c.6665C>A (p.Pro2222His) was found in a breast cancer patient with a strong family history of cancer. A case study of hereditary cancer syndrome was illustrated to highlight the importance of genetic testing. CONCLUSION: This is the first largest analysis of carrier frequency and mutation spectrum of HCS in Vietnam. The findings demonstrate the clinical significance of multigene panel testing to identify carriers and their at-risk relatives for better cancer surveillance and management strategies.

Shear stress associated with cardiopulmonary bypass induces expression of inflammatory cytokines and necroptosis in monocytes.

Cardiopulmonary bypass (CPB) is required during most cardiac surgeries. CBP drives systemic inflammation and multiorgan dysfunction that is especially severe in neonatal patients. Limited understanding of molecular mechanisms underlying CPB-associated inflammation presents a significant barrier to improve clinical outcomes. To better understand these clinical issues, we performed mRNA sequencing on total circulating leukocytes from neonatal patients undergoing CPB. Our data identify myeloid cells, particularly monocytes, as the major cell type driving transcriptional responses to CPB. Furthermore, IL-8 and TNF-α were inflammatory cytokines robustly upregulated in leukocytes from both patients and piglets exposed to CPB. To delineate the molecular mechanism, we exposed THP-1 human monocytic cells to CPB-like conditions, including artificial surfaces, high shear stress, and cooling/rewarming. Shear stress was found to drive cytokine upregulation via calcium-dependent signaling pathways. We also observed that a subpopulation of THP-1 cells died via TNF-α-mediated necroptosis, which we hypothesize contributes to post-CPB inflammation. Our study identifies a shear stress-modulated molecular mechanism that drives systemic inflammation in pediatric CPB patients. These are also the first data to our knowledge to demonstrate that shear stress causes necroptosis. Finally, we observe that calcium and TNF-α signaling are potentially novel targets to ameliorate post-CPB inflammation.

Physiological profile of undifferentiated bovine blastocyst-derived trophoblasts.

Trophectoderm of blastocysts mediate early events in fetal-maternal communication, enabling implantation and establishment of a functional placenta. Inadequate or impaired developmental events linked to trophoblasts directly impact early embryo survival and successful implantation during a crucial period that corresponds with high incidence of pregnancy losses in dairy cows. As yet, the molecular basis of bovine trophectoderm development and signaling towards initiation of implantation remains poorly understood. In this study, we developed methods for culturing undifferentiated bovine blastocyst-derived trophoblasts and used both transcriptomics and proteomics in early colonies to categorize and elucidate their functional characteristics. A total of 9270 transcripts and 1418 proteins were identified and analyzed based on absolute abundance. We profiled an extensive list of growth factors, cytokines and other relevant factors that can effectively influence paracrine communication in the uterine microenvironment. Functional categorization and analysis revealed novel information on structural organization, extracellular matrix composition, cell junction and adhesion components, transcription networks, and metabolic preferences. Our data showcase the fundamental physiology of bovine trophectoderm and indicate hallmarks of the self-renewing undifferentiated state akin to trophoblast stem cells described in other species. Functional features uncovered are essential for understanding early events in bovine pregnancy towards initiation of implantation.

Transcriptome profiling reveals activation of inflammation and apoptosis in the neonatal striatum after deep hypothermic circulatory arrest.

OBJECTIVES: Brain injury, leading to long-term neurodevelopmental deficits, is a major complication in neonates undergoing cardiac surgeries. Because the striatum is one of the most vulnerable brain regions, we used mRNA sequencing to unbiasedly identify transcriptional changes in the striatum after cardiopulmonary bypass and associated deep hypothermic circulatory arrest. METHODS: Piglets were subjected to cardiopulmonary bypass with deep hypothermic circulatory arrest at 18°C for 30 minutes and then recovered for 6 hours. mRNA sequencing was performed to compare changes in gene expression between the striatums of sham control and deep hypothermic circulatory arrest brains. RESULTS: We found 124 significantly upregulated genes and 74 significantly downregulated genes in the striatums of the deep hypothermic circulatory arrest group compared with the sham controls. Pathway enrichment analysis demonstrated that inflammation and apoptosis were the strongest pathways activated after surgery. Chemokines CXCL9, CXCL10, and CCL2 were the top upregulated genes with 32.4-fold, 22.2-fold, and 17.6-fold increased expression, respectively, in the deep hypothermic circulatory arrest group compared with sham controls. Concomitantly, genes involved in cell proliferation, cell-cell adhesion, and structural integrity were significantly downregulated in the deep hypothermic circulatory arrest group. Analysis of promoter regions of all upregulated genes revealed over-representation of nuclear factor-kB transcription factor binding sites. CONCLUSIONS: Our study provides a comprehensive view of global transcriptional changes in the striatum after deep hypothermic circulatory arrest and found strong activation of both inflammatory and apoptotic signaling pathways in the deep hypothermic circulatory arrest group. Nuclear factor-kB, a key driver of inflammation, appears to be an upstream regulator of the majority of the upregulated genes; hence, nuclear factor-kB inhibitors could potentially be tested for beneficial effects on neurologic outcome.

Metabolomic characteristics of cholesterol-induced non-obese nonalcoholic fatty liver disease in mice.

Nonalcoholic fatty liver disease (NAFLD) in non-obese patients remains a clinical condition with unclear etiology and pathogenesis. Using a metabolomics approach in a mouse model that recapitulates almost all the characteristic features of non-obese NAFLD, we aimed to advance mechanistic understanding of this disorder. Mice fed high fat, high cholesterol, cholate (HFHCC) diet for three weeks consistently developed hepatic pathology similar to NAFLD and nonalcoholic steatohepatitis (NASH) without changes to body weight or fat pad weights. Gas- and liquid chromatography/mass spectrometry-based profiling of lipidomic and primary metabolism changes in the liver and plasma revealed that systemic mechanisms leading to steatosis and hepatitis in this non-obese NAFLD model were driven by a combination of effects directed by elevated free cholesterol, cholesterol esters and cholic acid, and associated changes to metabolism of sphingomyelins and phosphatidylcholines. These results demonstrate that mechanisms underlying cholesterol-induced non-obese NAFLD are distinct from NAFLD occurring as a consequence of metabolic syndrome. In addition, this investigation provides one of the first metabolite reference profiles for interpreting effects of dietary and hepatic cholesterol in human non-obese NAFLD/NASH patients.

A brief history of the search for the protein(s) involved in the acute regulation of steroidogenesis.

The synthesis of steroid hormones occurs in specific cells and tissues in the body in response to trophic hormones and other signals. In order to synthesize steroids de novo, cholesterol, the precursor of all steroid hormones, must be mobilized from cellular stores to the inner mitochondrial membrane (IMM) to be converted into the first steroid formed, pregnenolone. This delivery of cholesterol to the IMM is the rate-limiting step in this process, and has long been known to require the rapid synthesis of a new protein(s) in response to stimulation. Although several possibilities for this protein have arisen over the past few decades, most of the recent attention to fill this role has centered on the candidacies of the proteins the Translocator Protein (TSPO) and the Steroidogenic Acute Regulatory Protein (StAR). In this review, the process of regulating steroidogenesis is briefly described, the characteristics of the candidate proteins and the data supporting their candidacies summarized, and some recent findings that propose a serious challenge for the role of TSPO in this process are discussed.

Current status and future perspectives: TSPO in steroid neuroendocrinology.

The mitochondrial translocator protein (TSPO), previously known as the peripheral benzodiazepine receptor (PBR), has received significant attention both as a diagnostic biomarker and as a therapeutic target for different neuronal disease pathologies. Recently, its functional basis believed to be mediating mitochondrial cholesterol import for steroid hormone production has been refuted by studies examining both in vivo and in vitro genetic Tspo-deficient models. As a result, there now exists a fundamental gap in the understanding of TSPO function in the nervous system, and its putative pharmacology in neurosteroid production. In this review, we discuss several recent findings in steroidogenic cells that are in direct contradiction to previous studies, and necessitate a re-examination of the purported role for TSPO in de novo neurosteroid biosynthesis. We critically examine the pharmacological effects of different TSPO-binding drugs with particular focus on studies that measure neurosteroid levels. We highlight the basis of key misconceptions regarding TSPO that continue to pervade the literature, and the need for interpretation with caution to avoid negative impacts. We also summarize the emerging perspectives that point to new directions that need to be investigated for understanding the molecular function of TSPO, only after which the true potential of this therapeutic target in medicine may be realized.

Translocator Protein (TSPO) Affects Mitochondrial Fatty Acid Oxidation in Steroidogenic Cells.

Translocator protein (TSPO), also known as the peripheral benzodiazepine receptor, is a highly conserved outer mitochondrial membrane protein present in specific subpopulations of cells within different tissues. In recent studies, the presumptive model depicting mammalian TSPO as a critical cholesterol transporter for steroidogenesis has been refuted by studies examining effects of Tspo gene deletion in vivo and in vitro, biochemical testing of TSPO cholesterol transport function, and specificity of TSPO-mediated pharmacological responses. Nevertheless, high TSPO expression in steroid-producing cells seemed to indicate an alternate function for this protein in steroidogenic mitochondria. To seek an explanation, we used CRISPR/Cas9-mediated TSPO knockout steroidogenic MA-10 Leydig cell (MA-10:TspoΔ/Δ) clones to examine changes to core mitochondrial functions resulting from TSPO deficiency. We observed that 1) MA-10:TspoΔ/Δ cells had a shift in substrate utilization for energy production from glucose to fatty acids with significantly higher mitochondrial fatty acid oxidation (FAO), and increased reactive oxygen species production; and 2) oxygen consumption rate, mitochondrial membrane potential, and proton leak were not different between MA-10:TspoΔ/Δ and MA-10:Tspo+/+ control cells. Consistent with this finding, TSPO-deficient adrenal glands from global TSPO knockout (Tspo(-/-)) mice also showed up-regulation of genes involved in FAO compared with the TSPO floxed (Tspo(fl/fl)) controls. These results demonstrate the first experimental evidence that TSPO can affect mitochondrial energy homeostasis through modulation of FAO, a function that appears to be consistent with high levels of TSPO expression observed in cell types active in lipid storage/metabolism.

Mitochondrial Translocator Protein (TSPO) Function Is Not Essential for Heme Biosynthesis.

Function of the mammalian translocator protein (TSPO; previously known as the peripheral benzodiazepine receptor) remains unclear because its presumed role in steroidogenesis and mitochondrial permeability transition established using pharmacological methods has been refuted in recent genetic studies. Protoporphyrin IX (PPIX) is considered a conserved endogenous ligand for TSPO. In bacteria, TSPO was identified to regulate tetrapyrrole metabolism and chemical catalysis of PPIX in the presence of light, and in vertebrates, TSPO function has been linked to porphyrin transport and heme biosynthesis. Positive correlation between high TSPO expression in cancer cells and susceptibility to photodynamic therapy based on their increased ability to convert the precursor 5-aminolevulinic acid (ALA) to PPIX appeared to reinforce this mechanism. In this study, we used TSPO knock-out (Tspo(-/-)) mice, primary cells, and different tumor cell lines to examine the role of TSPO in erythropoiesis, heme levels, PPIX biosynthesis, phototoxic cell death, and mitochondrial bioenergetic homeostasis. In contrast to expectations, our results demonstrate that TSPO deficiency does not adversely affect erythropoiesis, heme biosynthesis, bioconversion of ALA to PPIX, and porphyrin-mediated phototoxic cell death. TSPO expression levels in cancer cells do not correlate with their ability to convert ALA to PPIX. In fibroblasts, we observed that TSPO deficiency decreased the oxygen consumption rate and mitochondrial membrane potential (ΔΨm) indicative of a cellular metabolic shift, without a negative impact on porphyrin biosynthetic capability. Based on these findings, we conclude that mammalian TSPO does not have a critical physiological function related to PPIX and heme biosynthesis.

Minireview: translocator protein (TSPO) and steroidogenesis: a reappraisal.

The 18-kDa translocator protein (TSPO), also known as the peripheral benzodiazepine receptor, is a transmembrane protein in the outer mitochondrial membrane. TSPO has long been described as being indispensable for mitochondrial cholesterol import that is essential for steroid hormone production. In contrast to this initial proposition, recent experiments reexamining TSPO function have demonstrated that it is not involved in steroidogenesis. This fundamental change has forced a reexamination of the functional interpretations made for TSPO that broadly impacts both basic and clinical research across multiple fields. In this minireview, we recapitulate the key studies from 25 years of TSPO research and concurrently examine their limitations that perhaps led towards the incorrect association of TSPO and steroid hormone production. Although this shift in understanding raises new questions regarding the molecular function of TSPO, these recent developments are poised to have a significant positive impact for research progress in steroid endocrinology.