Glioblastoma (GBM) is an immunosuppressive, lethal brain tumor. Despite advances in molecular understanding and therapies, the clinical benefits have remained limited, and the life expectancy of patients with GBM has only been extended to ~15 months. Currently, genetically modified oncolytic viruses (OV) that express immunomodulatory transgenes constitute a research hot spot in the field of glioma treatment. An oncolytic virus is designed to selectively target, infect, and replicate in tumor cells while sparing normal tissues. Moreover, many studies have shown therapeutic advantages, and recent clinical trials have demonstrated the safety and efficacy of their usage. However, the therapeutic efficacy of oncolytic viruses alone is limited, while oncolytic viruses expressing immunomodulatory transgenes are more potent inducers of immunity and enhance immune cell-mediated antitumor immune responses in GBM. An increasing number of basic studies on oncolytic viruses encoding immunomodulatory transgene therapy for malignant gliomas have yielded beneficial outcomes. Oncolytic viruses that are armed with immunomodulatory transgenes remain promising as a therapy against malignant gliomas and will undoubtedly provide new insights into possible clinical uses or strategies. In this review, we summarize the research advances related to oncolytic viruses that express immunomodulatory transgenes, as well as potential treatment pitfalls in patients with malignant gliomas. Oncolytic virus-encoded immunomodulatory transgene therapy for gliomas has yielded beneficial outcomes.Oncolytic virus and tumor-targeting immune modulatory therapies have shown synergistic inhibition of malignant gliomas.Oncolytic virus immunotherapy of malignant gliomas has been used in clinical trials.The combination of stem cell carriers with oncolytic virus therapy for gliomas enhances antitumor efficacy.Can the immune system attack and engulf exogenous viruses?Are glioma stem cells resistant to viral therapy?Can the presence of nontumor cells such as tumor stroma cells impede the spread of oncolytic viruses?In personalized medicine, should potential challenges be considered for the treatment of patients with malignant gliomas?2. Current standard treatment options for malignant gliomas are multimodal and include surgical resection, postoperative radiotherapy, and concomitant chemotherapy with temozolomide4. Due to the invasive growth and recurrence features of malignant gliomas, the prognosis for patients with malignant gliomas remains extremely poor, with a median survival of nearly 15 months for newly diagnosed patients6. Thus, more specific, safe, and efficient treatment strategies are required. A growing body of preclinical and clinical data suggest that genetically engineered oncolytic viruses may be effective therapeutic agents used in the treatment of malignant gliomas.Glioblastoma (GBM) is both the most aggressive and lethal malignant brain tumor in adults and accounts for more than 30% of intracranial tumors8, and the underlying mechanism may involve OV-induced cell destruction by cancer-specific genetic alteration, as well as sequential virus release and viral infection. It was first reported that tumors could be inhibited or shrunk in patients with cervical cancer and rabies virus positivity9. Researchers found that a conditionally and genetically modified replication-competent oncolytic virus was selectively toxic to tumor cells and nontoxic to normal cells12. Cassel et al. reported that a genetically engineered oncolytic virus was evaluated as an adjunctive therapeutic agent for patients with malignant melanoma13. Subsequently, oncolytic viruses, including G207 and HSV1716, were used for clinical research in patients with malignant gliomas in the USA and the UK14. In 2015, the FDA approved the use of an oncolytic virus in the USA to treat patients with melanoma15. In addition, recent advances in viral therapy, of which the most promising are perhaps viruses such as DNX-2401, PVS-RIPO, and Toca 511, have shown complete durable responses in ~20% of GBM patients who received virus intratumorally18. However, the clinical trial did not meet its primary endpoints. These encouraging results obtained with PVS-RIPO, Toca 511, and DNX-2401 have been granted a fast track designation by the FDA for expedited drug review processes.Oncolytic virus (OV) therapy is a novel and promising therapeutic approach for tumors that involves selectively infecting and killing tumor cells. Prior research studies demonstrated that αvβ3 integrin and nectin-1 are required for efficient infection of cells by herpesvirus and adenovirus, respectively19. Furthermore, Friedman et al. summarized the milestones of oncolytic viruses carrying exogenous genes for cancer treatment20. In 2014, we also demonstrated that in animal models of human GSCs, an oncolytic HSV-1 that encodes an endostatin–angiostatin fusion gene could greatly enhance antitumor efficacy compared to HSV-1 without the fusion gene by generating antitumor angiogenic activity21. Later, our research team found that viruses that express the suicide gene cytosine deaminase (CD) could significantly enhance antitumor efficacy and prolong the life expectancy of tumor-bearing animals by the subsequent conversion of nontoxic prodrugs into toxic prodrugs23. Our research team independently developed a novel oHSV-1 containing the CD therapeutic gene. Moreover, a clinical trial using an engineered oHSV-1 (ON-01) injected intratumorally into patients with recurrent or refractory intracranial malignant gliomas is currently ongoing at Beijing Tiantan Hospital .Our research team previously found that the Endo–Angio fusion gene that is expressed in glioma stem cells (GSCs) could be administered via infection by oncolytic HSV-1, which carries an exogenous Endo–Angio fusion gene24. Moreover, oncolytic virus-mediated destruction of the tumor tissues was closely related to robust stimulation of innate antiviral immune responses and adaptive antitumor T-cell responses25. To further augment the antitumor effects, certain research studies have transduced immunomodulatory therapeutic transgenes, such as IL-1528, IL-1232, IL-434, and TRAIL36, via oncolytic virus, which can effectively inhibit or kill tumor cells through the antitumor immune response. Therefore, oncolytic viruses that encode immunomodulatory transgenes are gradually becoming a research hot spot in the field of glioma treatment. The gene required for oncolytic virus growth is typically placed under the control of a tumor-specific promoter or enhancer. In 2019, Yan et al. constructed a novel recombinant oncolytic adenovirus that expresses IL-15 and under the control of the E2F-1 promoter; it was found that this novel adenovirus could selectively kill tumor cells and exhibit increased antitumor effects both in vitro and in vivo26. Moreover, oncolytic viruses that were armed with immunomodulatory transgenes have been evaluated in several preclinical and clinical trials for the treatment of malignant gliomas. Genetically engineered virotherapy for malignant gliomas has so far been proven safe and effective. Nevertheless, many innovative strategies are currently under development to improve intratumoral viral expansion and antitumor efficacy without compromising security. Overall, previous studies on genetically engineered virotherapy with immunomodulatory transgenes have resulted in breakthroughs regarding several difficulties, which may contribute toward new insights into future therapeutics for patients with malignant gliomas.A preclinical study demonstrated that intratumoral administration of OVs could drive the development of systemic antitumor immunity and result in elimination of contralateral tumorsOncolytic viruses are a distinct class of antitumor agents with a unique mechanism of action that selectively replicate and target tumor cells, including GBM cells, affecting the tumor while sparing normal tissues and generating beneficial outcomes in GBM patients. However, diverse factors restrict the effect of virus treatment alone. In recent years, previous studies have inserted immunomodulatory genes into the viral genome, enabling the virus to release immune factors and simultaneously kill tumor cells in a direct manner; further, the antitumor immunologic reaction has been optimized, which has become a new approach for antitumor therapy. Oncolytic viruses that express immunomodulatory transgenes have been evaluated in terms of efficacy and safety in several preclinical and clinical trials for the treatment of malignant gliomas. However, their therapeutic utility in GBM appears to be limited due to several challenges.This review provides a summary of ongoing studies on engineered oncolytic viruses that express immunomodulatory transgenes for the treatment of malignant gliomas, as shown in Table 38. In addition, the antiangiogenic properties of IL-12 have been identified and characterized41, which may represent a second potential mechanism for its antitumor activity. In recent years, researchers found that oncolytic viruses that express IL-12 could powerfully produce an antiglioma immune response in a glioma-bearing model31. Thus, the potential mechanisms of IL-12-mediated antitumor activity depend not only on the activation of the innate and adaptive effector immune systems but also on the inhibition of angiogenesis.IL-12 is a heterodimeric protein that plays a pivotal role in linking the innate and adaptive immune systems. Previous data have demonstrated that IL-12 is a cytokine with potent antitumor properties. It is produced by antigen-presenting cells, including B lymphocytes, dendritic cells, and monocytes. It serves to augment the cytolytic activities of NK cells and cytotoxic T lymphocytes and enables the development of a TH-1-type immune response46. Moreover, the safety and efficacy of the oncolytic virus were evaluated, and some strides were made in a clinical study. In 2017, Saha et al. found that the combination of an oncolytic HSV that expresses IL-12 with immune checkpoint inhibitors, including anti-CTLA-4 and anti-PD-1 antibodies, could potently eradicate glioma cells and extend the survival of glioma-bearing mouse models29. This combination provides new insight into glioma therapy. In brief, previous data and preclinical safety studies of another oncolytic HSV that expresses IL-12 also provide a compelling rationale for the clinical translation of IL-12-armed oncolytic HSVs for the treatment of patients with malignant gliomas.Based on the antitumor activity and antiangiogenic properties of IL-12, genes that express IL-12 have been inserted into the engineered oncolytic virus genome. In 2013, many studies reported that a genetically engineered oncolytic herpes simplex virus, when armed with the immunomodulatory cytokine interleukin 12, significantly enhanced the survival of tumor-bearing mice and effectively inhibited tumor growth47. TRAIL is a strong therapeutic candidate for the treatment of glioblastoma because TRAIL can potently induce tumor-specific apoptosis48. With a deeper understanding of glioma therapy, TRAIL is widely used to exploit new therapeutic strategies. Most recent data have shown more potent antitumor efficacy of oncolytic viruses that encode TRAIL than of oncolytic viruses without TRAIL expression in xenograft models of subcutaneous and orthotopic glioblastoma, specifically through superior induction of apoptosis and the promotion of extensive viral distribution in tumor tissues49. Based on the properties of TRAIL-induced apoptosis of tumor cells, oncolytic viruses that express TRAIL could provide interesting approaches for glioma treatment.One novel strategy for tumor treatment is to induce apoptosis of tumor cells. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) superfamily and can induce apoptosis of tumor cells through activation of the TNF/CD95L axis and spare the majority of nonmalignant cells52. In 2017, Rivera et al. found that IL-15 could decrease the migration, invasion, and proliferation of tumor cells and inhibit angiogenesis53. Moreover, oncolytic viruses that express IL-15 significantly lyse tumor cells and reduce the tumor volume by activating natural killer (NK) cells, CD8+ T cells, and other immune cells54. Furthermore, our research team found that the destruction capacity of oncolytic adenovirus armed with IL-15 on tumor cells was stronger than that of the control virus (data not shown). Thus, the above evidence indicates that oncolytic virus-encoded IL-15 generates antitumor efficacy by activating innate and adaptive effector immune mechanisms, as well as by inhibiting angiogenic activity. This opens a new therapeutic direction for patients with malignant gliomas. However, due to limited research on oncolytic viruses encoding IL-15 for glioma treatment, further studies are necessary.IL-15, which is a part of the 4a-helix bundle cytokine family, is a crucial factor in immune regulation and primarily comes into play through the activation of NK cells. It is a receptor complex that consists of three subunits: a unique a-chain, a b-chain (shared with IL-2), and a common g-chain. As a novel molecular agent, IL-15 is used in tumor research and shows powerful antitumor immune responses through the stimulation of natural killer cells29. Immune checkpoint blockers can regulate different inhibitory pathways on immune cells, overcome the immunosuppressive microenvironment, and further promote antitumor immunity through distinct and nonredundant immune evasion mechanisms55. Previous studies have demonstrated therapeutic efficacy with immune checkpoint inhibitors in orthotopic glioma-bearing models29. Moreover, Lukas et al. demonstrated the safety and efficacy of an immune checkpoint blocker (anti-PD-L1 antibody) in patients with recurrent GBM in a clinical trial56. In recent research, Passaro et al. confirmed that oHSV-1 encoding an anti-PD-1 antibody showed potent and durable antitumor immune responses after intratumoral administration and improved survival in preclinical GBM models57. Thus, OVs armed with immune checkpoint inhibitors may be a promising strategy for GBM patients in the future.Immune checkpoints, especially PD-1, PD-L1, and CTLA-4, are immunosuppressive molecules that are upregulated in the GBM microenvironment58, which makes it a good option for OV therapy to increase activation of T cells, which further recognize antigens on tumor cells infected with the virus. Its mechanism is due to tumor-specific immunity, and this modality is more tumor-specific than immune checkpoint blockade. Previously, an oncolytic adenovirus expressing the immune costimulator OX40L exhibited superior tumor-specific activation of lymphocytes and proliferation of CD8+ T cells specific to tumor-associated antigens, resulting in cancer-specific immunity59. In addition, granulocyte macrophage colony-stimulating factor (GM-CSF) is also a potent inducer of tumor-specific, long-lasting antitumor immunity in both animal models and human clinical trials9. Prior work demonstrated that oncolytic viruses expressing GM-CSF produced stronger antitumor immune responses in human solid tumors than oncolytic viruses not expressing GM-CSF60. Recent studies have investigated the efficacy and safety of oncolytic viruses encoding GM-CSF and their potentially enhanced immune activity in glioma-bearing models62. This tumor-specific strategy will be beneficial and efficacious for glioma patients in the future.OX40 ligand (OX40L) is an immune costimulator that binds the unique costimulator OX40 on T cells63. Given its deletion and mutation in tumors, some studies have transfected the PTEN gene into glioma cells, and the expression of exogenous PTEN impeded the growth of glioma via augmentation of the immune response. Previous research revealed that recombinant adenovirus armed with the PTEN gene was able to inhibit the proliferation and tumorigenicity of glioma cells64. In addition, P53 is also a tumor suppressor gene involved in several aspects of cell cycle control and suppression of transformation. P53-armed virus significantly decreased tumor volume and prolonged the survival of mice compared with control virus in GBM models66. A recombinant adenovirus expressing p53 in glioma cells led to biological effects of the newly expressed p53 protein, which induced apoptosis to produce rapid and generalized death of human glioma cells67. Therefore, using recombinant viruses for the delivery of tumor suppressor genes into glioma cells is a promising, rational, and effective approach to treat glioma based on the transfer of genes.Phosphatase and tensin homolog (PTEN) is recognized as a candidate tumor suppressor gene based on the presence of inactivating mutations in several tumors68. Recent studies have shown that oncolytic herpes viruses expressing E-cadherin inhibit the clearance of NK cells to enhance viral spread and cause tumor regression, and oHSVs encoding E-cad remarkably prolongs the survival rate in GBM-bearing mouse models69. This suggests that E-cad can be added as a module to oncolytic viruses to improve therapeutic efficacy in GBM.E-cadherin (E-cad) is a calcium-dependent cell–cell adhesion molecule that cooperates with nectin-1 in the formation of cell–cell adherent junctions. E-cad binding to the receptor KLRG1 protects E-cad-expressing cells from being lysed by NK cells. Overexpression of E-cad on virus-infected cells may directly facilitate cell-to-cell infection, which is the dominant method of intratumoral viral spread70. In 2006, Curtin et al. investigated an adenoviral vector expressing Flt3L (AdFlt3L) that induced a specific increase in the levels of IFN-α secreted by DCs in the brain71. Moreover, AdFlt3L prominently suppressed glioma growth and improved survival when tumors were treated within 3 days of implantation in an intracranial glioma model by creating an inflammatory environment72. In addition, Barnard et al. found that oHSVs expressing Flt3L significantly extended life expectancy in glioma-bearing mice73. This is a novel approach in which virus-mediated expression of Flt3L leads to either eradication of the tumor or significant extension of the life span of animals.FMS-like tyrosine kinase 3 ligand (Flt3L) stimulates maturation and proliferation of DCs and NK cells. Flt3L protein could retard tumor progression and decrease the number of tumor metastasesAlthough oncolytic viruses that are armed with immunomodulatory transgenes for malignant glioma treatment have become increasingly popular, all oncolytic virus-based therapies, like other therapies, appear to be limited in their effectiveness. For example, the host responds to the oncolytic virus by inducing intratumoral infiltration of macrophages that can engulf the virus, limiting the potential of this therapeutic strategy. Some previous studies on the therapeutic effects of oncolytic viruses encoding immunomodulatory transgenes for malignant glioma treatment revealed potential pitfalls.78, as shown in Fig. 83. Over many decades, research groups have shown that oncolytic viruses can modulate changes in the TME and further generate antitumor immunity87. However, the regulatory mechanism of oncolytic viruses remains unclear, and adverse consequences of TME alteration have been gradually explored and reported.The tumor microenvironment (TME) is increasingly recognized as an important determinant of tumor progression and therapeutic resistance. A thorough understanding of the properties and functions of the TME is required to obtain a complete understanding of brain tumor biology and treatment. Generally, the tumor microenvironment consists of tumor cells, fibroblasts, endothelial cells, tumor stem cells, mesenchymal stem cells (MSCs), tumor-associated macrophages (TAMs), the extracellular matrix, and microglia, as well as cytokines and chemokines that are secreted by tumor and stromal cells92, which may be a pitfall of immunomodulatory therapy, as shown in Fig. 94, macrophages can engulf and eliminate virus particles, which results in a significant reduction in virus titer in tumor sites. Recent studies have demonstrated that TAMs and microglia can limit the replication and spread of oncolytic viruses89. Myeloid-derived suppressor cells (MDSCs) are known to act as major immunosuppressive cells within the GBM microenvironment. MDSCs induce B cell-mediated immunosuppression, perhaps inhibiting the effective response to oncolytic viruses armed with immunomodulatory transgenes95. In addition, the blood–brain barrier (BBB) limits the delivery of viral vectors, greatly compromising their oncolytic efficacy25. Moreover, related investigations have shown that GSCs and glioma-associated mesenchymal stem cells (Gb-MSCs) in the TME are likely to generate resistance to oncolytic virus therapy96. Therefore, the number of effective viruses that arrive at the targeted sites will be greatly reduced. In 2017, Pulluri et al. reported that TME changes could generate resistance to immune checkpoint inhibitors in metastatic melanoma97, which indicates the possibility of cytotoxic properties in the glioma microenvironment. Last, these data raise controversial issues regarding the association between the TME and oncolytic viruses in terms of benefits or harm for efficacy. Thus far, there has not been a conclusive finding98. The previously mentioned pitfalls must be seriously considered, and potential mechanisms need to be further exploited. These data strongly suggest that the tumor microenvironment does pose potential safety hazards in terms of oncolytic virotherapy for malignant gliomas.It is also important to seek understanding of how oncolytic viruses are affected by the TME in terms of differences between the TME and the natural milieu of the viruses. With a further understanding of the TME, researchers have demonstrated that immune infiltrating cells in the GBM microenvironment not only attack exogenous viruses but also restrict their spread to tumor cells and surrounding sites99. The mechanisms that underlie enhanced angiogenesis are not precisely known, but preclinical data regarding herpetic stromal keratitis in wild-type HSV-1 infection revealed that angiogenesis may be induced by paracrine effects that result from the release of VP22 or CpG motifs in the HSV-1 DNA, which are required for viral replication101. However, previous studies demonstrated that oncolytic viruses could disrupt tumor vascular endothelial cells and inhibit angiogenesis by directly infecting and killing vascular endothelial cells104. Moreover, our research team found that the tube segment lengths of HUVECs treated with oncolytic adenovirus were significantly shorter than those of the controls. The potential mechanism may involve the oncolytic virus inhibiting tumor angiogenesis by targeting VEGF104. In addition, researchers have also reported that TRAIL significantly enhanced the angiogenic activity and migration ability of human microvascular endothelial cells in vitro and in vivo106. Additional data suggest that TRAIL primarily inhibits angiogenesis by inducing vascular endothelial apoptosis, which leads to vessel recession110. Moreover, IL-15 and IL-12 could significantly decrease the number of blood vessels, which generates antiangiogenic effects53. Nevertheless, there is apparently no definitive conclusion for whether oncolytic viruses that express immunomodulatory transgenes support or suppress tumor angiogenesis.Angiogenesis, the process by which new blood vessels develop from the preexisting vascular endothelium, plays a crucial role in solid tumor recurrence and progression. Hence, antitumor vascular therapy has also become a popular approach for malignant gliomas. However, it is worth mentioning that there is no definite conclusion on whether oncolytic viruses that express immunomodulatory transgenes support or suppress tumor angiogenesis. These confusing issues are interesting for researchers and the basis of several promising studies. In 2012, Sahin et al. found that oncolytic viral therapy with the oncolytic virus HF10 contributed to tumor angiogenesis and thus supported tumor growth; further, this may possibly have been due to the inflammatory response that was induced by viral infection and viral proteins that were expressed during viral replicationTo avoid further confusion, these pitfalls must be thoroughly studied and resolved. With potential breakthroughs, a new era will begin for patients with malignant gliomas. Oncolytic virus-encoded immune cytokines have shown different abilities to promote or suppress tumor angiogenesis under different conditions. If these oncolytic viruses expressing immunomodulatory transgenes were investigated as glioma treatments, the effects of their support or suppression of the angiogenic potential of gliomas should also be considered.+ T cells, and regulate cytokine production and memory T-cell survival and proliferation54. Thus, these complex elements could largely induce the apoptosis and lysis of tumor cells. Despite generating antitumor effects, the immune system also attacks the virus, which results in limited potency and expansion. This may explain why viruses cannot reach and kill distant tumors effectively.Genetically engineered oncolytic viruses that are injected into the host can stimulate the immune system, generating a dual curative effect, which includes virus treatment and gene therapy. Research has shown that immune regulatory factors, including IL-4, IL-15, and IL-12, can enhance the activity of macrophages and neutrophils, contribute to the production and activation of natural killer cells and CD8113, as shown in Fig. 89. In 2015, Kober et al. found that microglia and astrocytes that were recruited in the TME preferentially cleared viral particles by immediate uptake after delivery, thus not allowing efficient viral infection114. As such, the efficacy and safety of genetically engineered oncolytic viruses must be guaranteed before wide application in clinical practice.Previous studies revealed that intravascularly injected viruses could be neutralized and eliminated by antibodies in the host115. Data from multiple studies demonstrate that higher levels of intratumoral heterogeneity predispose patients to inferior responses to anticancer therapies116. Heterogeneity provides a novel understanding of therapeutic resistance; thus, an accurate assessment of tumor heterogeneity is necessary to develop effective therapies.Tumor heterogeneity is one of the most common characteristics of malignant tumors that contributes to tumor progression and recurrence117. Previous studies using single-cell analysis have identified the great heterogeneity in different GBM subtypes and emphasized the clinical significance of GBM heterogeneity119. The TME and location of the tumor can influence intratumoral heterogeneity, while different molecular subtypes, such as the proneural and classical mesenchymal subtypes, influence intertumoral heterogeneity. In 2014, Reardon et al. revealed that tumor heterogeneity significantly influenced the outcomes of patients with glioblastoma115. Tumor heterogeneity makes tumors highly resistant to different therapeutic strategies, which results in decreased efficacy122. One possible mechanism of resistance involves the heterogeneous expression of the epidermal growth factor receptor (EGFR) in GBM123. This poses a substantial challenge for the effective use of EGFR-targeted therapies. In addition, our research team has repeatedly found that the sensitivity of different glioma cell lines to genetically engineered oncolytic adenoviruses is discrepant in research (data not shown), which suggests powerful treatment challenges for patients with malignant gliomas. Therefore, designing targeted therapies based on a range of molecular profiles can be a more effective strategy for eradicating treatment resistance, recurrence, and metastasis. Despite harvesting remarkable results in clinical trials, the therapeutic effects of oncolytic viruses on different types of malignant gliomas may be incredibly diverse. These data provide researchers with different insights into clinical treatment. Thus, a better understanding of glioblastoma heterogeneity is crucial for promoting therapeutic efficacy.It is well known that intertumoral and intratumoral heterogeneity persist in GBM, which is complicated due to the diverse tumor cell origins that are shown in glioma-bearing models and clinical tumor specimens127. However, one related study verified that the effect of oncolytic virus immunotherapy combined with anti-PD-1 antibody in glioblastoma was significantly better than that of oncolytic virus immunotherapy alone125. Thus, this combination should be translated to the clinic and tested against other immunosuppressive cancers.Oncolytic virotherapy is a promising approach in which viruses are genetically modified to selectively replicate in tumor cells. Furthermore, the efficiency and safety of these viruses have been demonstrated in preclinical and clinical studies. Due to the previously mentioned obstacles, the spread and concentration of oncolytic viruses in the TME are restricted. To overcome such obstacles, a better understanding of the TME and better designed strategies for improved therapeutic efficacy should be pursued in research on oncolytic viruses for malignant glioma treatment. Oncolytic viruses encoding immunomodulatory transgene therapy for malignant gliomas may become a popular approach that is widely used for clinical practice in the future. Furthermore, the immune checkpoint molecule known as PD-1 has become a research hot spot in tumor research. For cancers with an immunologically ‘cold’ TME , immune checkpoint blockade immunotherapy alone has not yet been successful128. In 2008, Sonabend et al. reported that administration in MSCs enabled oncolytic virus delivery to distant glioma cells and that this delivery significantly enhanced the infection and apoptosis of tumor cells compared to injection alone, revealing a therapeutic advantage129. Moreover, in prior investigations, MSCs carrying oncolytic viruses were injected into the carotid artery of mice, and these cells migrated to tumor sites, which resulted in inhibited glioma growth and improved survival of glioma-bearing animals132. Since then, many studies using neural stem cells loaded with oncolytic adenoviruses have demonstrated extended delivery of the oncolytic virus and prolonged survival of glioma-bearing animals that were treated with the stem cell-mediated oncolytic virotherapy135. The given data reveal that stem cell vectors could improve the outcomes of oncolytic virotherapy for glioma treatment. With ongoing exploration of trans-differentiation techniques, the barrier of sourcing could be overcome. The advantages of combining stem cell vehicles with oncolytic virus-encoding immunomodulatory transgene therapy for patients with malignant gliomas may be a greater area of focus in the next few decades.The current approach for oncolytic virus administration mainly includes direct intratumoral injections and systemic vascular delivery. Stereotactic injections or resection bed inoculation are the most commonly studied and simplest methods for introducing viral vectors into high-grade gliomas. This approach has the advantage of bypassing the BBB and introduces a high concentration of viruses directly into the tumor tissues. However, it is limited by multiple factors in the host and only delivers a single dose. Currently, stem cell-based therapy for gliomas has emerged as a promising novel strategy. Given their inherent tumor-tropic migratory properties, stem cells can serve as vehicles for the delivery of therapeutic efficacyGenetically engineered oncolytic virus therapy for malignant gliomas has recently been an increasing focus of research. Oncolytic viruses that express immunomodulatory transgenes generate antitumor effects via oncolytic and immunotherapy effects. Despite the numerous oncolytic virus-related studies that have produced great strides in the field of glioma treatment, serious obstacles remain. Moreover, side effects that are caused by virotherapy in clinical trials exist. It remains uncertain whether oncolytic viruses, once appropriately modified, can be used to treat different types of gliomas. These issues need to be further explored and explained in future studies. In conclusion, using oncolytic viruses that are armed with immunomodulatory transgenes for malignant glioma treatment is still in its early stages, and a better understanding of the biological consequences of this therapy is required before its wide use in treating patients with malignant gliomas.


Genetic quality assurance (QA), including genetic monitoring (GeMo) of inbredstrains and background characterization (BC) of genetically altered (GA) animalmodels, should be an essential component of any QA programme in laboratoryanimal facilities. Genetic quality control is as important for ensuring thevalidity of the animal model as health and microbiology monitoring are. Itshould be required that studies using laboratory rodents, mainly mice and rats,utilize genetically defined animals. This paper, presented by the FELASA WorkingGroup on Genetic Quality Assurance and Genetic Monitoring of Laboratory Murines,describes the objectives of and available methods for genetic QA programmes inrodent facilities. The main goals of any genetic QA programme are: (a) to verifythe authenticity and uniformity of inbred stains and substrains, thus ensuring agenetically reliable colony maintenance; (b) to detect possible geneticcontamination; and (c) to precisely describe the genetic composition of GAlines. While this publication focuses mainly on mouse and rat genetic QA, theprinciples will apply to other rodent species some of which are brieflymentioned within the context of inbred and outbred stocks. Syngeneic animals will permanently accept tissue transplants from anyindividual of the same strain and sex. Unlike cloned animals and monozygotictwins , inbred rodents, besidesbeing isogenic, are also homozygous at almost all genomic loci. Overall, eachinbred strain represents a unique, although fortuitous, assortment of alleles.3 If a strain were to be remade from scratch, using the same founders,after the same 20 generations of inbreeding it would create a geneticallydistinct strain due to the random assortment and fixation of alleles. Baselinephenotypic data for the most common inbred mouse strains are available through acoordinated international effort initiated by The Jackson Laboratory andimplemented through The Mouse Phenome Database (http://phenome.jax.org/).4 An example of baseline phenotypic data is presented in SupplementaryTables 1A and 1B. The Mouse Genome Informatics (MGI) website5 provides a list, compiled by Dr Michael Festing ,of 420 inbred mouse and 230 inbred rat strains (some of which have been lost orterminated), along with brief descriptions. The list includes widely used inbredmouse strains: A/J, BALB/c, C3H/He, C57BL/6, DBA/2, FVB/N and others; and ratstrains: ACI, BN, F344, LE and WKY.Isogeneity implies 6 However, several geneticists have disputed this use and have criticizedstudies in which outbred mice were used inappropriately, wasting both animallives and precious resources in suboptimal experiments.7Outbred stocks are populations of laboratory animals that differ from inbredstrains in that they are genetically heterogeneous. Compared with inbred strainsor F1 hybrids, the genetic constitution of a given animal, taken randomly froman outbred stock, is not known a priori. However, all of the animals in thegroup share group characteristics (identity), such as being albino , good breeders and relatively tame comparedto other strains; features that make these animals very popular as fostermothers for assisted reproductive techniques. Examples of outbred stocks of miceare ICR (CD-1), CFW and NMRI  and (non-Swiss) CF-1. Examples ofoutbred rat stocks are Sprague Dawley (SD), Wistar (WI) and Long-Evans (LE).Since outbred stocks are not genetically defined, quality control is commonlybased on assessing expected phenotypic traits, such as coat colour, growth andreproductive characteristics, based on data from the large colonies ofcommercial breeders. Because outbred colonies, like human populations, areheterogeneous, they are frequently used in toxicology and pharmacology research.F1 hybrids result from the outcross of two separate inbredstrains and are heterozygous at all loci for which the parental strains harbourdifferent alleles. F1 littermates are genetically identical and arehistocompatible. Congenic strains are produced by crossing twostrains: the donor strain that carries the allele orchromosomal region of interest, and the recipient orbackground strain that will receive the locus of interest.F1 offspring generated by crossing donors and recipients are then backcrossed tothe recipient strain. Offspring that carry the allele of interest are identifiedand again backcrossed to the background strain. This process is typicallyrepeated for 10 or more successive generations  aims tocharacterize the gene alteration that is responsible for a specific mutant phenotype. Reversegenetics is the opposite approach and aims to characterize the functionof a gene by analysing the consequences (at the phenotypic level) of alterationsnormally engineered by researchers at the DNA level. This section introduces thefour basic types of GA rodents, those created by: (a) pronuclear microinjection, (b)vector- mediated transgenesis (c) homologous recombination in embryonic stem (ES)cells, (d) gene editing nucleases, and (e) either chemically induced or spontaneousmutations. Detailed descriptions of the technologies used to create GAs have been published.8 Before selecting a gene-editing technique to create a genetically modifiedanimal, it is important to check an appropriate database such as those hosted by TheJackson Laboratories and the International Mouse Phenotyping Consortium as towhether a suitable animal model already exists (see Supplementary Table 2 for thecomplete list of online resources for laboratory mouse and rat strains).Before presenting the different types of GA rodents, it is worth mentioning thatthere are basically two different approaches to characterizing gene function.9 and were the first transgenic animals. It is advisableto use the term ‘transgenic’ only for animals whose genomes have been altered bythe random insertion of DNA.  or genetically modifiedmice (GMM) are typically used to describe any type of genetic modification inthe mouse. We use the term GA rodent here to also include those carryingspontaneous or chemically induced mutations, and ‘line’ instead of ‘strain’ forGA rodents.) Transgenic rodents are almost exclusively created by the pronuclearmicroinjection of foreign DNA fragments directly into one of the two pronucleiof one-cell embryos (zygote), a technique that is still widely used. In thisprocess of additive transgenesis, the microinjected transgenerandomly integrates into the genome as a single copy or more often as aconcatemer with variable copy number. The mouse and rat models created with thissystem typically express or, in the resultant concatemer, overexpress atransgene placed under the control of a tissue-specific,developmental-stage-specific, or ubiquitous promoter , all contained in the transgene DNA construct.Transgenic mice were introduced in the early 1980s10 In order to achieve a pure genetic background (recommended), thetransgene must be introduced into embryos derived from an inbred strain.The recommended generic symbol for a transgenic insertion is Tg. The foundertransgenic animals are hemizygous for the DNA segment and are designated Tg/0.Transgenes are extra segments of DNA that have no corresponding ‘wild-type’sequence in the unmodified homologous chromosome in hemizygous animals, that iswhy the use of ‘0’ instead of ‘+’ is recommended. Each transgenic line generated via random integration creates aunique animal model and each putative founder must be developed independently.Traditionally, to distinguish between homozygous (Tg/Tg) and hemizygous (Tg/0)mice, the mouse of interest was crossed to a non-transgenic partner and theprogeny were statistically analysed for Mendelian segregation of the transgene.A more modern technique uses quantitative real-time polymerase chain reaction(qPCR) to distinguish hemizygous from homozygous transgenic mice.bigenic) mice.11A later improvement on the constructs used in the transgenesis approach was theintroduction of inducible systems in which transgene expression can be turned onand off. Examples of this strategy are the Tet-on and Tet-off expressionsystems. In these systems, transcription of a given transgene is placed underthe control of a tetracycline-controlled trans-activator protein, which can beregulated, both reversibly and quantitatively, by exposing the transgenic miceto either Tetracycline (Tc) or one of its derivatives, such as Doxycycline(Dox). Both Tet-on and Tet-off are binary systems that require the generation ofdouble transgenic (12 and transposons.13 Also pre-treated spermatozoa have been successfully used as vectors incombination with ICSI (intracytoplasmic sperm injection).14 Each technique has advantages and disadvantages and the correspondingprinciple of transgene integration may affect the quality of the resulting GAmodels. Viral vectors and transposons for instance integrate as a single copy,however multiple integrations, randomly distributed in the genome, are notuncommon. Major concerns exist regarding the impact of sperm-mediated genetransfer on the sperm genetic material, possibly induced by the pre-treatment of spermatozoa.15Alternative methods for transgenesis by random integration are based on vectorsof different origin. Most important and very efficient are retroviral/lentiviral vectorsAnother important technology utilizes murine ES cell lines. ES cells areundifferentiated, pluripotent, embryonic cells derived from the inner cell massof pre-implantation blastocysts that can participate in forming the germ-celllineage of chimeric mice, an indispensable step in generating founder micecarrying the targeted mutation. Historically, the first ES cell lines werederived from embryos of the 129 family , that is inbredstrains originally bred for the isolation of embryonic carcinoma (EC) cells.Today ES cell lines are available from many mouse strains and those of theC57BL/6N origin have become widespread and are often selected for trans-nationalprojects (e.g. EUCOMM).loxP system.16 Production of conditional KOs requires two independent lines: oneproviding a source of Cre recombinase, an enzyme derived from bacteriophage P1,in the tissue under study, and another containing loxP (locusof X-ing over P1) sitesflanking the DNA segment of interest that needs to be crossed to generate doublemutant mice. The Cre enzyme cuts and recombines the ‘floxed’DNA at loxP sites. The Cre transgene can be made inducible,adding more sophistication to the system. The tamoxifen-inducibleCreERT2 which can be activated in a spatio-temporal manner byadministration of tamoxifen, is widely used.17 The Cre-loxP strategy can also be used to regulate theexpression of reporter genes. For example, the lacZ gene can bedriven by a ubiquitous promoter (e.g. Rosa 26) with a floxedstop sequence, containing several terminator codons inserted between thepromoter and the lacZ coding sequence.In cases where constitutive null alleles lead to complex phenotypes, reducedviability, or have other drawbacks, conditional alleles may be used, allowingone to control the time and tissue where a gene is turned off, typically usingthe Cre/Over the last 10 years, a number of new techniques have been developed for theproduction of targeted mutations using engineered nucleases. These techniques,briefly described here, provide ES cell-independent methods to create targetedmutations in laboratory mice, rats and other species.FokInuclease domain must be designed. Each peptide is designed to recognize aspecific DNA sequence spanning 9–18 base pairs (bp) on either side of a 5–6 bpsequence, which defines the targeted region. When injected into a pronucleus orcytoplasm of zygotes, the ZFN assemblies bind tightly, one on each strand, onboth sides of the target site. The dimerized FokI endonucleasethen creates double strand DNA breaks (DSBs) triggering cellular mechanisms torepair the damage. Damage is normally repaired by either homology-directedrepair (HDR) or non- homologous end joining (NHEJ). HDR requires a homologoustemplate to guide the repair and thus re-establishes the original sequence. NHEJis much less precise and cause nucleotide deletions that lead to frame shiftsthat create potential loss-of-function or truncation mutations. Mice and ratscarrying null alleles or sequence-specific modifications have already beenproduced using ZFN technology.18 Like ZFNs, transcription activator-like effector nuclease (TALEN)technology involves the combination of a nonspecific DNA endonuclease fused to aDNA-binding domain, but can be more easily engineered (compared to ZFN) totarget a particular DNA sequence.To make mutations using zinc-finger nucleases (ZFN), two complementary andsequence-specific multi-finger peptides containing the 19 CRISPR-based guide RNAs (gRNAs) are designed to target a Cas endonucleaseto cut DNA at the desired site through RNA-guided DNA cleavage. The RNA-guidedendonucleases can be engineered to cleave virtually any DNA sequence byappropriately designing the gRNA, for example to generate KO mice.20 CRISPR/Cas technology has several advantages over ZFNs and TALENs. Themain advantage is the ease of design and the flexibility of using asequence-specific RNA interacting with the Cas enzyme instead of a complexsequence-specific protein (DNA-binding domain) fused to a nuclease. Also,mutations in multiple genes can be generated in a single step by injecting micewith multiple gRNAs that simultaneously target different genes.21 Such multiplex gene editing has been successful in cells, as well asmouse and rat embryos.20 CRISPR/Cas9 has been used to create insertions, deletions and pointmutations. The system is highly flexible, fast and efficient, and isrevolutionizing genomic engineering in mammals.22 It allows making KO and KI lines in any genetic background. DNA can beelectroporated (with size restrictions) or injected into either the cytoplasm orpronuclei of 1-cell or 2-cell stage embryos, thus avoiding the use of ES cellsand chimeras. However, as each engineered animal is unique, this technologyrequires extensive sequence analysis to characterize multiple putative foundersto ensure the presence of the desired mutation and the absence of undesired on-and off-target mutations or unpredictable larger genome alterations,24 while alsoidentifying mosaic founders (G0). Once identified, the selected founder shouldbe bred with wild-type animals to evaluate transmission of the mutation.The CRISPR /Cassystem, commonly implemented as CRISPR/Cas9, is based on a primitive defencemechanism that allows bacteria and archaea to fight against infection fromviruses, plasmids and phages.nude (Foxn1nu), scid (Prkdcscid), hairless (Hrhr), diabetes (Leprdb), obese (Lepob) and X-linked muscular dystrophy (Dmdmdx) in the mouse; and the mutations behind the Rowett nude(Foxn1rnu) and Zucker diabetic fatty (Leprfa) models in the rat.A list of GA rodent types is not complete without including both spontaneous andchemically-induced mutations. Spontaneous mutations, generally identifiedthrough the observation of an abnormal phenotype, present several advantages.First and foremost, they are produced at virtually no cost and are generallyfreely available. Second, they usually have an obvious phenotype, as they areidentified based on observation. Third, spontaneous mutations represent a greatvariety of molecular events, such as deletions, insertions and point mutations,generating not only loss-of-function alleles but also hypomorphic andhypermorphic alleles. Finally, mutations arise in a variety of backgroundsincluding inbred strains and outbred stocks. Several spontaneous mutations haveprovided rodent models for human conditions. These include classical mutationssuch as, 25The discovery of the extraordinary virtues of the alkylating agentN-ethyl-N-nitroso urea (ENU) as a mutagen was a milestone in the history ofmouse genetics. Researchers using ENU have generated and propagated numerousmutant alleles for protein-coding genes, thus establishing a precious tool forgenome annotation. Because ENU typically creates point mutations, it has beenwidely used in forward genetic screens. The major drawback of ENU-inducedmutagenesis is that it creates random mutations rather than targeted mutations.Several projects have been undertaken to systematically and extensivelyphenotype the offspring of ENU-mutagenized males. Large ENU mutagenesisprogrammes have been conducted in Germany, England and the USA.26The possibility of crossing different GA lines combined with the increasingcomplexity of targeting approaches has greatly increased the number ofavailable GA models. The need to cross different GA lines together for aparticular study generates additional complexity, especially at the geneticbackground level. Many mutants have been and are still generated on a hybridgenetic background. Therefore, it is essential to keep adequate records ofdetailed information for all genetically modified strains. This informationmust be transferred with the strain to all collaborators and users. The mostimportant information includes the correct strain name, a completedescription of the mutation, the genetic background of the animals, agenotyping protocol and observed phenotypic changes. Together, these providethe minimum information for the recommended ‘rodent-passport’, and severalforms have been designed for cataloguing this information. We recommend thedata sheet developed by the FELASA Working group on the refinement ofmethods for genotyping genetically modified rodents.27 A name designed according to the international nomenclature rules isthe only means to unambiguously distinguish strains from each other. This isimportant when the same strain is held in different facilities around theworld and/or they are listed in archives and databases. Further, it isimperative that strains be properly described in publications using auniversal nomenclature. Without a common nomenclature, it becomes impossibleto accurately communicate scientific results. Vague or incomplete namescreate errors rendering experiments irreproducible.Every mutant strain name must provide precise information on the affectedgene, the type of mutation and the genetic background. For in-housegenerated strains, one must provide a specific Institute for LaboratoryAnimal Research Laboratory (ILAR) Code Registration for the laboratory wherethe mutant originated. An overview on the importance of nomenclature can befound in the ‘FELASA guidelines for the production and nomenclature oftransgenic rodents’.de novo spontaneous mutations.A serious challenge facing rodent animal facilities is keeping inbred strainsgenetically pure and GA lines on a defined background. Changes in the geneticconstitution of inbred strains can be produced by (a) contamination by accidentaloutcrosses and (b) genetic drift due to residual heterozygosity or fixation ofTyrc/Tyrc), agouti (A/A), or non-agouti (a/a)).Where lines have the same coat colour alleles, extra care must be taken whenhousing them in close proximity of each other.The accidental mating of individuals from one inbred strain with animals ofanother origin is by far the most important source of genetic profile alterationin inbred strains. Genetic contamination of this type, which always results in asudden and massive exchange of alleles, is more likely between strains that havesimilar coat colour  at a locus among individuals, groups, orpopulations, at a frequency >1%. Two types of genetic markers are commonlyused in association studies and genetic quality control: microsatellites andsingle nucleotide polymorphisms (SNPs) (see ‘Marker systems’ below).Spontaneous mutations are a source of uncontrolled genetic variation that isoften impossible to detect by simple phenotypic observation or routine geneticmonitoring (GeMo). genetic drift. Genetic drift contributes inexorably tostrain divergence and the generation of substrains when the same strain ispropagated independently in different places.28 Examples of mouse substrains are abundant, for example there arec. 10 documented BALB/c substrains and c.15 C57BL/6 substrains including the J and N substrains from The JacksonLaboratory (Jax) and the National Institutes of Health (NIH), respectively.29 In the same way, many rat inbred strains present at least two substrains,for example SHR has at least four substrains (including SHR/Ola and SHR/NCrl),and WKY and F344 have at least three substrains each. Substrain variability hasbeen confirmed by sequencing analysis for these rat substrains,30 with WKY showing the highest degree of substrain variation (this is inpart due to the supply of the model prior to the prescribed 20 generationinbreeding requirement).While permanent inbreeding effectively eliminates a proportion of new mutantalleles, another undetected fraction may become progressively fixed in thehomozygous state, replacing the original allele, a process known aspassengermutations.31 There are many examples in the literature where substrains originatingfrom the same inbred strain have acquired new phenotypes as a consequence ofgenetic drift.32 For example, mice of the C57BL/6JOlaHsd substrain are homozygous for adeletion of the α-synuclein (Snca) and multimerin(Mnrn1) genes.34 Likewise, some spontaneousmutations differentially segregate in C57BL/6J and C57BL/6N, the most commonsubstrains of C57BL/6, separated in 1951. These include a retinal degenerationmutation in the Crb1 gene (Crb1rd8), present only in the N substrain, and a deletion in theNnt gene, present only in the J substrain.36 Berghe andcolleagues recently reported that passenger mutations are also common in most GAlines derived from 129 ES cells, and that these mutations persist even after thecreation of fully congenic strains.37 This is not trivial; Berghe et al. estimated that close to 1000protein-coding genes could be aberrantly expressed in the 129-derivedchromosomal segments that are still segregating in these congenic lines. Thisfinding emphasizes the need for properly chosen control animals to identifyphenotypes due to background mutations or the combination of backgroundmutations and the genetic modification of interest, rather than the modificationitself.Mutations that are hidden in the genomes of substrains or GA lines and can affectthe outcome of an experiment are sometimes referred to as http://www.informatics.jax.org/mgihome/nomen/strains.shtml). Ahelpful and visual Mouse Nomenclature Quick Guide is available at https://www.jax.org/jax-mice-and-services/customer-support/technical-support/genetics-and-nomenclature#.For more details on nomenclature refer to the Supplementary material.Rules guiding nomenclature were established by the International Committee onStandardized Genetic Nomenclature for Mice and Rats and are continuously updated.These rules, last revised in January 2016, are described on the MGI webpage under‘Guidelines for Nomenclature of Mouse and Rat Strains’ . A collection of mapped,highly polymorphic, SSLP markers for inbred laboratory rat strains is availablein The National BioResource Project – Rat database and is linked to the MapReport of the Rat Genome Database (RGD) (http://rgd.mcw.edu). SeeSupplementary Table 2 for the complete list of online resources for laboratorymouse and rat strains.Many polymorphisms have been described in the mouse and rat; however, onlymicrosatellites and SNPs are used as genetic markers in current QA programmes.Microsatellite markers, also known as Simple Sequence Length Polymorphisms(SSLPs) or Short Tandem Repeats (STRs), are still used in modern GeMo programmesbecause they are inexpensive and easy to type.c. 300 inbred, wild-derived, congenic, consomicand recombinant inbred strains maintained at The Jackson Laboratory.40 Several publications have reported useful SNPs for the rat. For example,Zimdahl and colleagues described a map with >12,000 gene-based SNPs fromtranscribed regions.41SNP genotyping is an alternative to microsatellites that is now widely used forGeMo. SNP genotyping is inexpensive and can be performed in most researchinstitutions or outsourced. SNPs are the most common genetic variation and existin both coding and non-coding regions. Almost all SNPs are bi-allelic,presenting one of only two possible nucleotides (e.g. homozygous G/G or T/T), orboth (e.g. heterozygous G/T) in an individual. Petkov and co-workers from TheJackson Laboratory have described the allelic distribution of 235 SNPs in 48mouse strains and selected a panel of 28 SNPs sufficient to characterize themajority of the 42 designed to effectively limit cumulative genetic drift by rebuildingtheir foundation stocks from pedigreed, cryopreserved embryos every fivegenerations. For example, starting in 2005, they began selling only C57BL/6Jmice derived from two chosen mice through hundreds of frozen embryos of theduo's grandchildren (enough to last for 25–30 years). It should be noted thatwhen recovering strains from frozen stocks good GeMo should be carried out toassure oneself that genetic contamination or wrong genotypes were not presentprior to freezing.Most GeMo techniques used currently are based on microsatellites or SNPs.However, GeMo should not rely solely on molecular techniques, but should take abroader view that includes phenotypic parameters such as coat colour, behaviourand breeding performance. Commercial breeders are extremely sensitized to therisk of genetic contamination and regularly monitor their strains for geneticcontamination, but not necessarily genetic drift, by using different sets ofSNPs to monitor their nucleus colonies. The Jackson Laboratory incorporated aunique, patented, Genetic Stability ProgramFor outbred stocks, GeMo helps preserve the genetic heterogeneity and allele poolof a colony. This complex process requires analysing a large number of animalsand comparing this data with historical data documenting the alleles present,their frequency and the level of heterozygosity in that particular colony. Insome cases, the results can reveal a loss of genetic variability resulting in acolony with very low heterogeneity. The degree of genetic heterogeneity inoutbred colonies depends on their history. Low heterogeneity can result frompoor selection of future breeding stock, deviation from approved breeding systems or the bottleneck effect caused by a small breeding pool, as iscommon when a small group of breeders is imported or being used to rederive acolony. In contrast, high heterogeneity can result from a recent outcross. Ingeneral, outbred stocks are characterized by measuring phenotypic traits andcalculating the corresponding mean and standard deviations. Essentially, geneticcontrol of outbred stocks is directed at avoiding inbreeding and stabilizinggenetic diversity over many generations.The best recommendation here is to purchase animals from reliable vendors andreplace the breeding stock with animals from the same vendor after 10generations, rather than to maintain independent colonies of classicalinbred strains. As an additional benefit, using animals from the same vendorprevents the formation of substrains harbouring potential mutations andmaintains a similar microbiome. Nevertheless, in-house colonies shouldalways be tested with a small set of informative microsatellite markers orSNPs to confirm integrity.Microsatellites can be used to verify that the animals in an inbredcolony are essentially pure, with no traces of genetic contamination.This is especially important in facilities that maintain strains withthe same coat colour in the same room, a particularly dangerous practiceespecially when not using individually ventilated cage (IVC) systems.Microsatellite testing can normally be performed in-house. The number ofmarkers to use for testing has not been standardized: each situation andfacility differs in how many and which strains are kept. Nonetheless, apanel of 40 polymorphic SSLPs, evenly distributed across the autosomes,will rule out recent genetic contamination, if the markers candistinguish among the strains being analysed. Supplementary Table 3presents a small set of mouse SSLPs that could be used to authenticatesome classical inbred strains.Interpreting SSLP data is straightforward. Because inbred animals areisogenic and homozygous, they will present only one band in theelectrophoresis gel, representing a single allele, when genotyped for aparticular SSLP. The presence of any heterozygosity, indicated by twobands, or bands that do not coincide with those of the strain controlDNA, should be considered as indicating potential strain contamination.How fre43 has the advantage that it can be automated using 96- or 384-wellplates and pipetting robots for the PCR reactions . Another option, real-time PCR (TaqMan®) technology, uses specificprimers coupled with a sequence-specific, fluorescent TaqMan probe, iseffective and easy to automate; however, the cost per individual assayis expensive compared with KASP assays, and requires a more costlyreal-time thermocycler. Finally, microarray-based SNP genotyping is nottypically used for small scale, in-house GeMo, but may be an option forvendors of inbred mice. When using or requesting microarray genotypingservices, be aware that only a percentage of the SNPs will bepolymorphic between the strains under analysis .Information regarding which alleles  to expect for aparticular SNP/strain combination, and their genomic location areavailable for hundreds of thousands of SNPs and for the common mouse andrat inbred strains in easily accessed databases and genome browsers(Supplementary Table 2).For GeMo purposes only, 40 polymorphic SNPs, evenly distributed acrossthe chromosomes is a reasonable number for detecting recent geneticcontamination (this suggestion should be modified dependent on theconditions or risks in each facility). SNP genotyping is currentlyavailable on different platforms, that vary in cost and automationcapabilities. Kompetitive Allele Specific PCR (KASP), a variation onallele-specific PCR, uses allele-specific oligo extension andfluorescence resonance energy transfer,44 One of the main problems of in-house outbred stocks is that they areoften maintained with a very small number of animals in the breeding colony,causing a reduction of alleles represented in the population. This mayimpact genetic drift and increase the inbreeding coefficient. Such coloniesare neither truly outbred nor inbred. Although SSLPs or SNPs can be used toestimate the level of heterozygosity within the colony, if it is notpossible to keep an appropriate number of breeders, it is better to purchaseoutbred rodents from vendors that maintain a very large colony and userecommended breeding schemes to reduce inbreeding.GeMo of outbred stocks is much more complex, because these animals are notgenetically uniform. Outbred colonies are essentially a group of closelyrelated animals, with shared ancestors and group identity, but that exhibitsome level of genetic heterozygosity. Since outbred colonies form apopulation rather than a strain, it is difficult to establish a standardGeMo programme with only a few genetic markers. However, with an adequatenumber of SNPs or SSLPs, allele frequencies within the population couldindicate the identity of the stock.46 One of the first cases reporting this phenomenon involvedthe classical diabetes (Leprdb) mutation that presented transient diabetes in a C57BL/6 background butovert diabetes in C57BLKS.47 Other examples include background effects on survival rate inEgfr  KO mice48 and tumour incidence in Pten KO mice.49 For this reason, every GA line should be characterized in terms of theirgenetic background. Moreover, the knowledge of the genetic background of amutation is also important for the selection of the corresponding control animals.50The explosion in the number of GA lines is exacerbating the problem of undefined‘mixed backgrounds’ in experimental rodents. This is particularly worrisome forinducible and conditional models that require the crossing of two independentlines (e.g. Cre-expressing lines crossed with floxed lines). Given that geneticbackground influences phenotype, especially through the influence of modifiergenes; mutations, both spontaneous and induced, transgenes, and targeted allelesthat are introgressed into a new background may not exhibit the expectedphenotype.51 whereas WT C57BL/6 females were typically used to prove germlinetransmission from the chimeras. Without subsequent backcrosses, this schemeresulted in a B6;129 mixed background. However, the availability of ES celllines derived from other strains (particularly from C57BL/6) and the arrival ofgenome editing techniques (section “Gene editing using nucleases”) that allowdirect production of targeted alterations in any mouse or rat strain52 is slowly changing this scenario. In any case, the problem of mixedbackground can be circumvented altogether by (a) injecting transgenes ornucleases (Cas9-sgRNA) into inbred embryos from the strain of choice; (b)modifying the gene of interest in ES cells from the preferred background strain(e.g. using C57BL/6 ES cells); and (c) crossing chimeras and KO/KI founders withmice of the same strain as the ES cells used for the targeting. Finally, if theGA line has already been developed or acquired from a collaborator orrepository, a BC should be performed, and if needed, a fully congenic strainshould be developed, either by classical or marker-assisted backcrossing.Periodic backcrossing of a congenic strain to the background strain (ofreputable source) also minimises divergence and keeps the congenic straingenetically close to the strain background of control animals.Genetic markers evenly distributed and covering the entire genome can be used ina genome scan to estimate the percentages of genome coming from different inbredorigins. This process of BC is provided by some commercial enterprises andinstitutional core facilities. A typical BC employs polymorphic markers todistinguish between the suspected inbred strains. In most mouse cases, thesestrains are C57BL/6 and 129 substrains because, historically, the ES cells usedfor the development of KO and KI mice through homologous recombination (section“Targeted mutagenesis by homologous recombination using ES cells” above) werederived exclusively from 129 substrainsmarker-assisted backcrossingor speed congenics.53 This process relies on using polymorphic genetic markers covering thewhole genome to determine the percentage of donor genome present in the animals,then selecting the animals with the lowest percentage of donor DNA for the nextbackcross to the recipient strain. This relies on the regions between thepolymorphic genetic markers being those of the donor genome: the denser thenumber of markers the higher the donor genome can be inferred. Common practiceis the use of 100–300 markers. This process reduces the number of generations toreach full congenicity (e.g. from N10 to N5), and therefore strain developmenttime, by approximately half. Using marker-assisted backcrosses and the rightnumber of animals we can obtain c. 80% recipient background atN2, c. 94% at N3, and c. 99% at N4 . A flowchart depicting astandard speed congenic protocol is shown in Figure 3. Ideally, the backcrossprocedure is started with a donor female and a recipient male. Then, F1 mutantmales will carry the correct Y-chromosome and after mating to a recipientfemale, males of the N2 generation will carry the correct X- and Y-chromosome ofthe recipient strain (avoiding the use of genetic markers on these chromosomes).54 It was predicted by Markel et al. that if 20 best breeders (carriers) areused at each generation of the speed congenics protocol >98% recipient genomecan be attained at N5.56 However, the chromosomal segments flanking the selectedlocus tend to remain associated with it and this is a limitation of the congeniclines due to the potential presence of modifier genes in this segments, theso-called ‘flanking gene problem’.57The use of DNA markers has allowed for a much more rapid and rigorous process ofcongenic strain development called 42 For outbred stocks, the intent is to minimize inbreeding, maintainheterozygosity and manage genetic drift that would otherwise lead to colonydivergence. Ideally, outbred colonies should be maintained with ≥25 breeding pairs,all of which have to contribute to the next generation, in order to avoid anincrease of the inbreeding coefficient per generation of more than 1%. Smallercolonies drift fast toward homozygosity because breeders are closely related.58For inbred, co-isogenic and congenic strains, breeding methods and genetic stabilityprogrammes help to minimize substrain divergence due to genetic drift, and also toprevent genetic contamination by accidental crosses with other strains. To reducegenetic drift, the number of generations of in-house breeding should be minimized,and the lines submitted to repositories such as, JAX, EMMA, MMRRC, IMSR or RIKEN, tobe archived as frozen embryos and/or sperm. This secures the line and provides ameans of replacing the breeding stock every 10 generations as recommended by TheJackson Laboratory Genetic Stability Program (GSP) in order to slow down cumulativegenetic drift.http://www.findmice.org/).Cryopreservation strategies have been adopted for long-term storage of embryos andgametes in several large centralized repositories including the EMMA/INFRAFRONTIER(European Mutant Mouse Archive), the Knock Out Mouse Project (KOMP) Repository, TheJackson Laboratory Repository, The Center for Animal Resources and Development(CARD) and the Riken Bio Resource Center, which can provide cryopreserved materialor live mice to laboratories. These repositories facilitate the availability ofthese strains to the worldwide scientific community and provide a backup for apotential loss of a strain. The International Mouse Strain Resource (IMSR) is asearchable online database of mouse strains, stocks and mutant ES cell linesavailable worldwide, including inbred, mutant and genetically engineered strains(Click here for additional data file.Supplemental Material for Genetic quality assurance and genetic monitoring oflaboratory mice and rats: FELASA Working Group Report by Fernando Benavides,Thomas Rülicke, Jan-Bas Prins, James Bussell, Ferdinando Scavizzi, PaoloCinelli, Yann Herault and Dirk Wedekind in Laboratory Animals


As many countries in Europe, France faced a second wave COVID-19 pandemic since September, 2020. During the first wave, intensivists faced an unprecedented massive admission of patients with COVID-19 pneumonia requiring invasive mechanical ventilation, sometimes leading to ICUs saturation. They discovered the stereotypical course of this previously unknown disease with its own specificities including the need for deep sedation and neuromuscular blockade, the increased risk of thrombotic and hemorrhagic events  with higOne can legitimately assume that the experience gained during the first wave may have contributed to a better management and outcome among critically ill COVID-19 patients admitted during the second wave.We therefore compared the characteristics and the outcome between patients admitted to our 41-bed COVID-19 ICU for acute respiratory failure due to COVID-19 (RT-PCR positive for SARS-CoV-2) during the first wave  and those admitted to our 18-bed ICU during the second wave .COVID-19 patients without acute respiratory failure, those transferred to other ICUs or still hospitalized in ICU were not included.Eighty-two patients were admitted during the first wave and 50 during the second wave. Comparison between first and second wave regarding patients’ characteristics, ICU scores, comorbidities, biological data, administered treatments and outcome is detailed in the Table p = 0.96) and duration of ICU stay did not differ between the two waves.Compared to the first wave, we observed a lower proportion of patients requiring invasive mechanical ventilation and a lower rate of thrombotic events. The delay between ICU admission and tracheal intubation was longer during the second wave. ICU mortality   Fig. .Fig. 1KaWe herein report the first study comparing the outcome of critically ill COVID-19 patients between first and second wave in France. Despite a better understanding of COVID-19 with significant treatment modification including systematic and early administration of glucocorticoids as well as intermediate/full dose thromboprophylaxis, we did not observe any decrease in ICU mortality, with still half of the patients dying in our ICU.However, patients admitted during the second wave were less likely to require invasive mechanical ventilation with a longer delay between ICU admission and tracheal intubation, potentially related to early administration of glucocorticoids . HoweverThe lower rate of thrombotic events observed during the second wave is likely inherent to the increased intensity of thromboprophylaxis even if a lower rate of CT pulmonary angiographies during the second wave cannot be ruled out.The strengths of our study include the similar typology and severity of the patients during both waves making relevant the comparison. Its monocenter design, implying similar criteria of ICU admission and similar criteria of tracheal intubation by the same team of intensivists during both periods, may also be considered as a strength.


Sus scrofa), using the calcaneus shape as a possible phenotypic marker. We first assessed differences in shape variation and covariation in captive-reared and wild-caught wild boars, taking into account differences in sex, body mass, available space for movement and muscle force. This plastic signal was then contrasted with the phenotypic changes induced by selective breeding in domestic pigs. We found that mobility reduction induces a plastic response beyond the shape variation of wild boars in their natural habitat, associated with a reduction in the range of locomotor behaviours and muscle loads. This plastic signal of captivity in the calcaneus shape differs from the main changes induced by selective breeding for larger muscle and earlier development that impacted the pigs' calcaneus shape in a much greater extent than the mobility reduction during the domestication process of their wild ancestors.Deciphering the plastic (non-heritable) changes induced by human control over wild animals in the archaeological record is challenging. We hypothesized that changes in locomotor behaviour in a wild ungulate due to mobility control could be quantified in the bone anatomy. To test this, we experimented with the effect of mobility reduction on the skeleton of wild boar ( Yet, To document the biological process of animal domestication, bioarchaeologists have relied on genetic markers of reproductive isolation and trait selection, or genetically induced morphological markers . These mIndependent of any genetic change, modifications in the use of a skeletal trait may trigger adjustments of the musculature, which can subsequently induce morphological variation through skeletal plasticity: muscle stimulation influences bone growth, development and remodelling ,28. To dSus scrofa), since ungulates played a major role as food in the Neolithic transition and because few studies have used ungulates to explore the effects of captivity [To test this hypothesis, we used an experimental approach on the wild boar  fenced forest in Urciers , where human interaction is intentionally kept to a minimum in order to ensure that the boars' behaviour remains as natural as possible. Consequently, the mobility of this control population is not very different from the pre-Neolithic situation in the Near East where wild boar were probably partly commensal before being fully domesticated [2 (0.3 ha) wooded pen and an indoor stall of 100 m2, where males and females were separated. To better control the effect of diet on the growth of the two groups, they were both supplied with standardized food pellets in order to maintain a healthy weight, according to the standard nutritional requirements of European wild boar populations [To experimentally test the plastic response of mobility reduction on the shape of the calcaneus in a wild ungulate, we rely on a control population of wild boar living in a 100 000 msticated ,41. Fromulations . Water wThis experiment received full ethical agreement (APAFIS#5353-201605111133847).2.2.The captive signal at adulthood in wild boars reared in stalls and pens was contrasted with 28 adult wild-caught wild boars from the control population in Urciers, three other populations in France and two in Switzerland . All theU nustrale) aged between 14 and 18 months. These pigs were bred according to the traditional extensive herding practice in Corsica where pigs can roam freely in large areas of maquis forest to access natural resources for their diet [To compare the plastic response of captivity with the phenotypic change driven by the last 200 years of artificial selection, we collected 19 domestic pigs, including 11 specimens from traditional landraces, which were part of a conservation programme, and eight from an intense breeding programme dedicated to industrial meat production . All theeir diet .2.3.® medical CT scanner with a spatial resolution of 100 to 500 micrometres at the CIRE imaging service of the INRA in Nouzilly. Sixteen specimens from Germany were scanned using the medical CT scanner of the Halle hospital with the same parameters. For each calcaneus, three-dimensional surfaces were obtained from the DICOM images stacks using Avizo v. 8.0.To compare the specimens from the different institutions, we combined three-dimensional images acquired from medical CT scan and photogrammetry. Previous studies have shown that the results from these methods are comparable ,45. SeveThe three-dimensional models of 13 specimens from Switzerland and MHK were obtained using photogrammetry. We used a Sony DSLR-A350 camera with a 50 mm lens. Each calcaneus was placed on a rigid, planar cardboard sheet with a calibrated reference pattern. The photographs were shot at regular intervals of approximately 22° near the proximal and distal extremities and 45° near the lateral and medial sides as the operator rotated the cardboard sheet. Sets of 12 pictures were acquired from three different vertical angles  for both dorsal and plantar sides. Some additional pictures were shot to ensure every detail was captured. In total, more than 72 (12 × 3 × 2) pictures were used to reconstruct each model. Sets of images were processed with Agisoft PhotoScan to obtain three-dimensional surface files of each calcaneus and scaled using the ‘Transform: Scale’ tool in MeshLab.2.4.To capture the complexity of the calcaneus form, encompassing the articulation and muscle insertion areas, we used a three-dimensional sliding semi-landmark procedure  figure . This in2.5.To analyse the covariation between life-history traits and the calcaneus form in wild boar, we collected body mass, sex and age for the wild boar specimens . Captive-raised wild boars have a known age at death, but wild-caught wild boars had to be aged according to their dental eruption and occlusal attrition stages ,51.2.6.−3) [To measure the covariation between calcaneus shape and the functional properties of the muscles attached to the calcaneus, we dissected the lateral gastrocnemius (LG), the medial gastrocnemius (MG) and the soleus (S) of 22 experimental captive-reared wild boars, four specimens from the control population, and eight wild boars from French populations (−3) , we calc−3) .2.7.In order to measure the influence of mobility restriction on calcaneus shape variation in wild boars, we collected information on the available area for each of the experimental groups and the wild populations . The areas available for the experimental contexts  were known; however, the size of enclosures for the MHK wild boars had to be estimated from archive photos. The available areas for free wild boar populations from France and Switzerland were measured using Google maps, based on the measured area of the forest in which the specimens were caught.2.8.2.8.1.The difference in calcaneus size and body mass variation among captive-reared and wild-caught wild boars, taking into account their sex, was visualized with a box plot and tested with factorial analysis of variance (ANOVA) and pairwise test. The correlation between calcaneus size and body mass was tested using the Pearson test.We tested whether the calcaneus shape differed among wild-caught and captive-reared wild boars using their status (GP1) as grouping factor while accounting for shape covarying with age, body mass and bone centroid size  using a factorial MANCOVA with 1000 permutations.2) and intrinsic muscle force (PCSA), we used partial least squares (PLS) analyses [We visualized and tested the PCSA differences among wild-caught and captive-reared wild boars using a box plot and ANOVA. The correlations between PCSA and calcaneus centroid size and body mass were tested with a regression and the Pearson test. For the visualization of shape deformation associated with body mass, calcaneus centroid size, available area for locomotion  and visualized with a box plot. The shape differences were tested with a Procrustes ANOVA and visualized using a Canonical Variate Analysis (CVA); both were performed on a shape-reduced dataset after a principal component analysis (PCA) performed on the Procrustes coordinates to keep 95% of the variance . To visuTo test the shape difference depending on the main categories  and their sex, including the interaction of these two factors, we used a factorial MANOVA with a 1000 permutations procedure.All the statistics were performed using R . Factori3.3.1.F = 1.171, p = 0.33) and calcanaeum centroid size  between the wild-caught and captive-reared wild boars. A slight trend towards greater bone size and body mass in captive-reared specimens compared to wild boars can be observed. Yet, no difference exists among the two captive-reared groups  but not in body mass . This absence of sexual dimorphism in body mass was mainly true for captive-reared specimens in stalls (b). The correlation between calcaneus centroid size and body mass was overall very strong . Yet, stall-reared specimens display weaker correlation than wild-caught and pen-reared specimens (c).We found no significant differences in body mass  as well as age effect over shape , body mass  and CS . The three groups of wild-caught and captive-reared wild boars show a common shape covariation with age  and centroid size , but differed in the way body mass impact the calcaneus shape .Shape differences were significant between wild-caught and captive-reared wild boars  and medial  gastrocnemius, but not for the soleus . Sexual dimorphism in muscle PCSA among wild-caught and captive-reared wild boars was significant only for the lateral gastrocnemius .Compared to the wild-caught wild boars, the captive-reared wild boars had significantly greater muscle PCSA for the lateral . Changes are localized in the distal part of the calcaneus with a more dorso-plantarily curved calcaneus and a shift of sustentaculum tali towards the distal extremity. The mobility reduction (b) and a greater force generation potential of the lateral and medial gastrocnemius muscles (d) is associated with a more elongated epiphysis that is curved towards the dorsal side and a more distally shifted sustentaculum tali.Similar calcaneus shape changes were found in association with body mass and calcaneus centroid size increase a,c. Chaneduction b and a g muscles d is asso3.2.F = 5.75, p < 0.001). All the domestic pigs from the traditional and improved breeds had a larger calcaneus than wild-caught and captive-reared wild boars, except for the Corsican free-ranging pigs which were intermediate and showed no significant differences with the landraces, improved breeds or the wild-caught and captive-reared wild boars. The German captive-reared wild boars were in the upper range of wild-caught wild boars, probably because they are old specimens and their different genetic pool. These results suggest that calcaneus size increase is not affected by a change in locomotor behaviour but rather by artificial selection.The calcaneus CS  differedF = 3.0564; p < 0.0001). The main shape differentiation shown on the first canonical axis (a) was driven by the divergence between wild boars and pig breeds, with modern breeds being the most divergent in shape. Factorial MANOVA showed that difference between wild boars/traditional pig breeds/modern breeds accounted for 19% of the total variance of calcaneus shape in adults (p < 0.01), while sexual dimorphism had almost no effect on shape variation and was similar in both wild boars and domestic breeds . The shape deformation associated with the divergence between wild-caught wild boars and domestic pigs  is marked by an increase in calcaneus robustness and thickness with a medio-laterally wider calcaneus, particularly on the medial side at the junction with the epiphysis where we can see a bulge. The coracoid process and the cuboid facet are also more robust and the epiphysis is longer in domestic pigs than in wild boars.The calcaneus shape differed significantly among the eight groups of wild boars and domestic pigs . The captive-reared wild boars were strongly divergent from their wild-caught relatives along this axis, whereas the difference between the stall- and pen-reared wild boars was not significant . In pigs, the free-roaming Corsican pigs are the most divergent from the captive, modern breeds. The shape divergence on CV2 (c) and along the vector between wild-caught and captive-reared boars mean shapes (d) was localized on the proximal epiphysis, which was longer and curved towards the dorsal side in captive specimens. The sustentaculum tali was also slightly shifted towards the distal end compared to wild-caught wild boars.The second canonical axis is related to the difference in mobility, expressed both in wild boars and pigs a. The cae on CV2 c and alon shapes d was loc4.4.1.Mus musculus) [Previous studies have shown that several generations of captivity-induced changes in diet, exercise and stress could impact the morphology and physiology of captive populations . Howeverusculus) .Mustela nigripes) after 10 years of captivity with increased mobility reduction over time [Indeed, we found no bone size reduction in captive-reared wild boars, suggesting that ankle bone shape has greater plasticity than overall bone size when a wild animal is facing modifications of its locomotor environment during its growth. Skeleton size reduction has been evidenced in captive black-footed ferrets  ,65, whico 150 cm  and dailo 150 cm . Compareo 150 cm . Reorieno 150 cm . Additio4.3.These plastic shape differences in captive-reared wild boars follow a different phenotypic trajectory compared to the shape divergence between wild-caught and domestic phenotypes induced by the last 200 years of selective pig breeding. However, this signal is also expressed in pigs, since we found a divergence between captive-reared and free-ranging pig breeds, suggesting that this plastic signal is of a more general nature. The patterns of covariation we observed between calcaneus shape and body mass, bone centroid size, available space for movements and muscle force suggest that shape divergence between domestic breeds and wild boar phenotypes may have been driven by increases in overall body mass and associated bone size . Selecti5.Our results provide strong evidence that changes in locomotor behaviour induced by captivity can impact the bone anatomy of a wild ungulate over the course of its lifetime. This plastic imprint of captivity in wild boar calcaneus produces a phenotype beyond the usual reaction norm in natural environments but along a different trajectory than the divergence driven by selective breeding. If we consider that human control and modification of a wild animal's movements is one of the first steps towards domestication, our results provide new methodological perspectives for bioarchaeological approaches linking plastic responses to the domestication process involving cultural control of wild animal movements. Further studies now need to explore when these changes are implemented during the growth to understand the developmental processes behind these plastic changes.


Diagnostic imaging of early-stage cholangiocarcinoma is challenging. A previous in vitro study of fixed-tissue liver resection specimens investigated T2 mapping as a method of exploiting the locally increased signal-to-noise ratio (SNR) of duodenoscope coils for improved quantitative magnetic resonance imaging (MRI), despite their non-uniform sensitivity. This work applies similar methods to unfixed liver specimens using catheter-based receivers.Ex vivo intraductal MRI and T2 mapping were carried out at 3T on unfixed resection specimens obtained from cholangiocarcinoma patients immediately after surgery using a catheter coil based on a thin-film magneto-inductive waveguide, inserted directly into an intrahepatic duct.Polypoid intraductal cholangiocarcinoma was imaged using fast spin-echo sequences. High-resolution T2 maps were extracted by fitting of data obtained at different echo times to mono-exponential models, and disease-induced changes were correlated with histopathology. An increase in T2 was found compared with fixed specimens and differences in T2 allowed the resolution of tumour tissue and malignant features such as polypoid morphology.Despite their limited field of view, useful data can be obtained using catheter coils, and T2 mapping offers an effective method of exploiting their local SNR advantage without the need for image correction. O. viverrini, widespread in Lao People’s Democratic Republic, Cambodia, central Vietnam and Myanmar;C. sinensis, occurs in South-West China, North Vietnam and Korea.Cholangiocarcinoma (CCA) is an adenocarcinoma of the biliary tree and the second most common primary malignancy of the liver.Local dishes often involve uncooked or pickled river fish. Following consumption, metacercariae excyst in the duodenum, pass through the ampulla of Vater into the bile ducts, where they attach to mucosa and develop. Their toxic excretions induce chronic irritation and hyperplasia of the ductal epithelium, oxidative DNA damage and possible malignant transformation.7The symptoms of early-stage CCA are minor  However, obstructive jaundice may occur as intraductal tumours develop. Biomarkers are still relatively ineffective,10Confirmatory diagnosis is performed during endoscopic retrograde cholangiopancreatography (ERCP),17All MRI suffers from motion artifacts24In vitro work with fixed specimensAn ex vivo intraductal imaging study was performed on resection specimens from Thai patients with CCA at Khon Kaen University Hospital (KKUH). The study was conducted in accordance with the Declaration of Helsinki, following the grant of ethics approval by the local Ethics Committee and informed patient consent. Careful synchronization between surgery, radiology and pathology in a busy hospital was needed to ensure fresh specimens. The patients selected were therefore three individuals adjacent on the surgery list in a 1-week period, with no exclusions. Specimens were made available after resection, imaged for around 2 hrs, and transferred to pathology. Pre-operative MRI was used to develop an imaging protocol. Due to the lack of perfusion, contrast agents could not be used.TM, Philips Healthcare, Best, Netherlands) at KKUH, using fast spin-echo (FSE) sequences. The quadrature body coil (QBC) was used for excitation. Signal reception was carried out using i) the QBC and ii) an experimental biliary catheter coil. Axial images obtained using the internal coil  were compensated using a self-developed Matlab algorithm. T2 maps were also generated using Matlab, which fitted mono-exponential relaxation models to image data obtained at different echo times; similar results were obtained by least-squares fitting to log-domain data.Imaging was carried out with a 3T clinical whole-body system  was operable, but the resection specimen could not be cannulated due to common bile duct obstruction. The best results were obtained from the third patient, a 70-year-old male. Dilation of the left lobe intrahepatic ducts was identified using ultrasound, and the cause was confirmed as intraductal tumour using MRCP.The upper photograph in The lower photograph in The specimen was located at the magnet isocentre with the catheter approximately axial, and localizers were acquired to establish the imaging volume. Five sets of axial images were acquired, each in a stack of 21 slices in the locations marked in To highlight the structure of the duct, The ex vivo data presented here suggest that there may be a role for catheter coils in improving diagnostic MRI of CCA using a modified ERCP procedure, since the need for particular coil orientations and slice planes may be obviated using T2 mapping. This modality seems most appropriate for early-stage intraductal and periductal tumours. Provided the duct may be cannulated, its tract may be deduced from the catheter track and the extent of the tumour from differences in T2 with respect to parenchyma. Despite these encouraging results, further steps must be taken before in vivo study.First, the local SNR advantage of internal receivers must be shown to be retained with the longer sequences used for relaxometry, even in the presence of respiratory motion.Second, clinical advantages must be demonstrated. Of particular interest would be the ability to distinguish the involvement of arteries and veins in the hilar region, since this often determines operability.Magnetic resonance images and T2 maps of intraductal CCA have been obtained in vitro following left hepatectomy, using a catheter-mounted thin-film receiver inserted directly into an intrahepatic duct in an unfixed resection specimen. The results confirm that T2 maps can provide useful visualization from images acquired using internal coils without the need for radial image correction. Due to the large difference in their T2 values, ducts and tumour boundaries can be separated from parenchyma without contrast agent, and details of duct walls are apparent. However, new forms of image presentation may be required for effective visualization. The method appears most appropriate for imaging of single ducts with early-stage disease, but work is needed to establish the viability and potential advantages of in vivo use.