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Marina Simón,1,* Jesper Tranekjær Jørgensen,1,* Kamilla Norregaard,1 Jonas Rosager Henriksen,2 Gael Clergeaud,2 Thomas L Andresen,2 Anders Elias Hansen,2 Andreas Kjaer1 1Department of Clinical Physiology and Nuclear Medicine & Cluster for Molecular Imaging, Copenhagen University Hospital – Rigshospitalet & Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark; 2Department of Health Technology, Section for Biotherapeutic Engineering and Drug Targeting, Technical University of Denmark, Kongens Lyngby, Denmark*These authors contributed equally to this workCorrespondence: Andreas Kjaer, Department of Clinical Physiology and Nuclear Medicine & Cluster for Molecular Imaging, Copenhagen University Hospital – Rigshospitalet & Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark, Email akjaer@sund.ku.dkIntroduction: Traditional cancer treatments, such as chemotherapy, are often incapable of achieving complete responses as standalone therapies. Hence, current treatment strategies typically rely on a combination of several approaches. Nanoparticle-based photothermal therapy (PTT) is a technique used to kill cancer cells through localized, severe hyperthermia that has shown promise as an add-on treatment to multiple cancer therapies. Here, we evaluated whether the combination of gold nanoshell (NS)-based PTT and liposomal doxorubicin could improve outcome in a mouse model of colorectal cancer.Methods: First, NS-based PTT was performed on tumor-bearing mice. Radiolabeled liposomes were then injected at different timepoints to follow their accumulation in the tumor and determine the ideal injection time after PTT. In addition, fluorescent liposomes were used to observe the liposomal distribution in the tumor after PTT. Finally, we combined PTT and doxorubicin-loaded liposomes and studied the effect of the treatment strategy on the mice by following tumor growth and survival.Results: PTT significantly improved liposomal accumulation in the tumor, but only when the liposomes were injected immediately after the therapy. The liposomes accumulated mostly in regions adjacent to the ablated areas. When PTT was combined with liposomal doxorubicin, the mice experienced a slowdown in tumor growth and an improvement in survival.Conclusion: According to our preclinical study, NS-based PTT seems promising as an add-on treatment for liposomal chemotherapy and potentially other systemic therapies, and could be relevant for future application in a clinical setting.Graphical Abstract: Keywords: liposomes, PET imaging, chemotherapy, hyperthermia, gold nanoparticles

Betina Børresen,1 Anders Elias Hansen,2,3 Frederikke Petrine Fliedner,2 Jonas Rosager Henriksen,3 Dennis Ringkjøbing Elema,3,4 Malene Brandt-Larsen,5 Lotte Kellemann Kristensen,2– 6 Annemarie Thuri Kristensen,1– 6 Thomas Lars Andresen,3 Andreas Kjær2,5 1Department of Veterinary Clinical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C 1870, Denmark; 2Cluster for Molecular Imaging, Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen N 2200, Denmark; 3DTU Health Technology, Center for Nanomedicine and Theranostics, Technical University of Denmark, Lyngby, Kgs 2800, Denmark; 4DTU Health Technology, The Hevesy Laboratory, Center for Nuclear Technologies, Technical University of Denmark, Roskilde, 4000, Denmark; 5Department of Clinical Physiology, Nuclear Medicine and PET, Copenhagen University Hospital, Copenhagen Ø 2100, Denmark; 6Minerva Imaging, Copenhagen N 2200, DenmarkCorrespondence: Andreas KjærCluster for Molecular Imaging, Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, Copenhagen N 2200, DenmarkTel +45 35 32 75 04Fax +45 35 32 75 46Email akjaer@sund.ku.dkBackground: The accumulation of liposome encapsulated chemotherapy in solid cancers is dependent on the presence of the enhanced permeability and retention (EPR) effect. Positron emission tomography (PET) imaging with a liposome encapsulated radioisotope, such as liposome encapsulated Cu-64 (64Cu-liposome) may help to identify tumors with high liposome accumulation, and thereby stratify patients based on expected benefit from liposomal chemotherapy. However, intravenous administration of liposomes without a cytotoxic content is complicated by the accelerated blood clearance (ABC) phenomenon for succeeding therapeutic liposome dosing. Alternative markers for assessing the tumor’s EPR level are therefore warranted.Materials and Methods: To increase our understanding of EPR variations and to ultimately identify an alternative marker for the EPR effect, we investigated the correlation between 64Cu-liposome PET/CT (EPR effect) and 68Ga-RGD PET/CT (neoangiogenesis), 18F-FDG PET/CT (glycolysis), diffusion-weighted MRI (diffusivity) and interstitial fluid pressure in two experimental cancer models (CT26 and COLO 205).Results: 64Cu-liposome and 68Ga-RGD SUVmax displayed a significant moderate correlation, however, none of the other parameters evaluated displayed significant correlations. These results indicate that differences in neoangiogenesis may explain some EPR variability, however, as correlations were only moderate and not observed for SUVmean, 68Ga-RGD is probably insufficient to serve as a stand-alone surrogate marker for quantifying the EPR effect and stratifying patients.Keywords: EPR effect, liposome, positron emission tomography, neoangiogenesis

Esben Christensen,1–3 Jonas R Henriksen,2,4 Jesper T Jørgensen,3 Yasmine Amitay,5 Hilary Shmeeda,5 Alberto A Gabizon,5 Andreas Kjær,3 Thomas L Andresen,1,2 Anders E Hansen1–3 1Department of Micro- and Nanotechnology, DTU Nanotech, Technical University of Denmark, DK-2800 Kongens Lyngby, Denmark; 2Center for Nanomedicine and Theranostics, Technical University of Denmark, DK-2800 Kongens Lyngby, Denmark; 3Cluster for Molecular Imaging, Department of Biomedical Sciences and Department of Clinical Physiology, Nuclear Medicine & PET, University of Copenhagen and Rigshospitalet, DK-2200 & DK-2100, Copenhagen, Denmark; 4Department of Chemistry, DTU Chemistry, Technical University of Denmark, DK-2800 Kongens Lyngby, Denmark; 5Shaare Zedek Medical Center and Hebrew University – School of Medicine, Jerusalem, Israel Background: Active, ligand-mediated, targeting of functionalized liposomes to folate receptors (FRs) overexpressed on cancer cells could potentially improve drug delivery and specificity. Studies on folate-targeting liposomes (FTLs) have, however, yielded varying results and generally fail to display a clear benefit of FR targeting.Method: Tumor accumulating potential of FTLs and NTLs were investigated in a FR overexpressing xenograft model by positron emission tomography/computed tomography imaging.Results: Tumors displayed significantly lower activity of FTLs than NTLs. Furthermore, FTLs displayed worse circulating properties and increased liver-accumulation than NTLs. Conclusion: This study underlines that long-circulating properties of liposomes must be achieved to take advantage of EPR-dependent tumor accumulation which may be lost by functionalization. FR-functionalization negatively affected both tumor accumulation and circulation properties. Keywords: liposomes, folate, cancer, imaging, PET, EPR

Due to drastic societal changes in the last few decades, there has been an increasing need for counseling services in China. Specifically, Chinese college students are confronted with many developmental challenges due to changes in expectations following college. The purpose of this phenomenological study was to explore Chinese college students' perceptions of counseling services in China. The authors identified six essential themes which made up the essence of this study. They were roles of counselors, issues that brought students to counseling, students' expectations in counseling, training and requirements for counselors, credentials and characteristics of counselor supervisors, and future development of counseling in China. The authors discussed the themes, implications, and offered recommendations for further research. [ABSTRACT FROM AUTHOR]

The multiple heritage (i.e., multiracial, hiracial) population has been identified by the US Census Bureau as the fastest growing population in the US. With this growth, there is also the diversity of those in multiple heritage families and with multiple heritage backgrounds. In this article, the authors describe the challenges faced by this population, the individual identity development process, and ways of helping individuals at different developmental levels along with their families. There is also an introduction to the new Competencies for Counseling the Multiracial Population (Kenney et al., 2013). [ABSTRACT FROM AUTHOR]

Studies involving student ambassadors have demonstrated their significance in recruiting prospective students to universities and colleges but they have not included the perceptions of the student ambassadors themselves. The purpose of this phenomenological study was to describe the perceptions and experiences of students who served as student ambassadors for their educational institution. Fifteen student ambassadors provided individual descriptions of their perceptions of being an ambassador. Four themes provided a description of ambassadors' perceptions: (a) belonging, (b) personal growth, (c) diversity, and (d) recruitment. A discussion of the benefits of student ambassadors as leaders are presented in terms of their ability to recruit for the university and college as well as the impact being a leader has on the ambassadors themselves. [ABSTRACT FROM AUTHOR]

The goal of this multiple case study was to investigate straight counseling students’ perceptions of their preparation to work with GLBT clients. For this study, semi-structured interviews were conducted with six straight counselor education students from two universities. Pattern matching analysis revealed that students with significant exposure to GLBT individuals, as well as those who believed their training provided adequate preparation for GLBT counseling, were more likely to adopt a GLBT-affirmative identity and to experience greater self-efficacy about counseling with GLBT individuals. In addition, thematic analysis revealed seven themes of participants’ experiences that fell into four categories. Ideas for counselor educators to incorporate experiential learning into their programs are offered. Keywords: GLBT, Ally, Identity, Counselor Education, Counselor Trainee, Case Study, Qualitative Research. [ABSTRACT FROM AUTHOR]

Much of the success of high-risk behavior prevention programs rests with teachers who deliver the curriculum however; few studies have investigated teachers' perceptions of program implementation. The objective of this phenomenological study was to answer the question, "What are the experiences of teachers who are asked to be involved in the implementation process when their school adopts a multiple high-risk behavior prevention program"? Participants included 10 teachers at a local, private high school in the Southern United States. Five themes emerged: (a) lack of consistent historical effort, (b) need for program, (c) positive but tentative perceptions, (d) challenges with implementation, and (e) review of program counselor. The qualitative results identified factors that can promote or hinder success of the program. [ABSTRACT FROM AUTHOR]

The phenomenon of absent fathers is increasing in the Black community and insight from Black males raised in these households is needed to understand how they persevere and succeed. This study explored the life experiences of collegiate Black males raised in absent father homes. Five themes emerged, which included a male role model/mentor, a supportive mother, desire to achieve an education, respect for their fathers, and resilience. The authors discuss these themes and present implications for future studies focused on counselors working with Black males raised in absent father households. [ABSTRACT FROM AUTHOR]

The purpose of this phenomenological study was to identify potential culture-related stressors in intercultural marriages and uncover some of the conscious and unconscious strategies applied by couples to cope with these stressors. In-depth ethnographic interviews were conducted with five intercultural couples. Interview data were then analyzed using a phenomenological reduction process to synthesize the meanings and essences of participant experiences. Results revealed that the intercultural couples studied used similar coping strategies to manage marital stressors that might be attributed to or accentuated by cultural differences. Six primary coping strategies were identified: (a) gender-role flexibility; (b) humor; (c) cultural deference by one partner; (d) recognition of similarities; (e) cultural reframing or the development of blended values and expectations that redefined the intercultural relationship; and (f) a general appreciation for other cultures. These intercultural coping strategies were supported by prior empirical research in the fields of intercultural communication and marital counseling. Further research is needed to understand better the unique stressors and coping mechanisms experienced by intercultural couples. Practical considerations for therapists who counsel intercultural couples are discussed. [ABSTRACT FROM PUBLISHER]

We investigated the training of licensed professional counselors in Texas by focusing on how students receive expertise in specialty areas such as substance abuse, sexual concerns, domestic violence, play therapy, family therapy, and art and music therapy. A mail survey elicited responses from department chairs or program directors of community counseling programs. Results of the survey were analyzed both quantitatively and qualitatively and indicated that most participating programs offer coursework in specialty areas designed to prepare students to work with a wide variety of client issues in counseling. [ABSTRACT FROM AUTHOR]

Counselor educators were surveyed regarding their perceptions of a proposed increase in the required number of semester hours for community counselors from 48 hours to 60 hours. Twenty-four participants (49%) indicated a preference for keeping the current 48 semester hour requirement; whereas 25 participants (51%) chose the other given option of 60 hours. Participants were also asked about the advantages and disadvantages of both 48-hour and 60-hour counselor training programs and themes from these open-ended responses were coded and analyzed. Common themes and implications, and recommendations for future research, are presented. [ABSTRACT FROM AUTHOR]

The Multiple Heritage Couple Questionnaire (MHCQ) is a revision and extension of the Interracial Couple Questionnaire (ICQ). It is designed to help counseling professionals attend to crucial information that is often overlooked when working with multiple-heritage couples. [ABSTRACT FROM AUTHOR]

Counselors and future counselors face a variety of challenges as society becomes more diverse and laden with differing value systems. As the Code of Ethics and Standards of Practice (American Counseling Association, 1995) is changed to become more inclusive of the values held in a diverse society changes in counselor preparation to incorporate these changing values are necessary. The authors discuss how values and ethics inform counselor preparation and recommend the application of J. A. Banks's (1981, 1988) multicultural educational pedagogy to counselor preparation programs. [ABSTRACT FROM AUTHOR]

Therapeutic interventions with Biracial women and girls are proposed with an emphasis placed on racial identity development. The Black/White Biracial Identity Development Model is presented as the basis for developing therapeutic interventions. The model was created based on a qualitative research study with Black/White Biracial women. It is proposed that an understanding of Biracial identity development is necessary in order to develop effective interventions that can assist Biracial women and girls with racial identity difficulties. [ABSTRACT FROM AUTHOR]

Presents a questionnaire for investigations into interracial marriages. Specific sociocultural and family influences on relationship; Counselors' personal attitudes, beliefs and biases toward interracial relationships and families.

This article presents a case study of the perceptions of a White female married to an African American male. [ABSTRACT FROM AUTHOR]

School counselors face many challenges implementing a Comprehensive Developmental Guidance and Counseling Program ([CDGCP] Dahir, Burnham, & Stone, 2009). This case study explored the experiences of a program director's, an administrator's, and three school counselors' implementation of a statewide CDGCP at the elementary level in a large suburban school district in Southeast Texas. The themes of differences in knowledge of CDGCP, benefits, time constraints, role inconsistencies, and moving toward a fully implemented CDGCP emerged from participants' responses. Implications and recommendations for other districts school counseling programs are provided. [ABSTRACT FROM AUTHOR]

Cyanobacteria are photosynthetic organisms that play important roles in carbon cycling and are promising bioproduction chassis. Here, we isolate two novel cyanobacteria with 4.6Mbp genomes, UTEX 3221 and UTEX 3222, from a unique marine environment with naturally elevated CO₂. We describe complete genome sequences for both isolates and, focusing on UTEX 3222 due to its planktonic growth in liquid, characterize biotechnologically relevant growth and biomass characteristics. UTEX 3222 outpaces other fast-growing model strains on a solid medium. It can double every 2.35 hours in a liquid medium and grows to high density (>31 g/L biomass dry weight) in batch culture, nearly double that of Synechococcus sp. PCC 11901, whose high-density growth was recently reported. In addition, UTEX 3222 sinks readily, settling more quickly than other fast-growing strains, suggesting favorable economics of harvesting UTEX 3222 biomass. These traits may make UTEX 3222 a compelling choice for marine carbon dioxide removal (CDR) and photosynthetic bioproduction from CO₂. Overall, we find that bio-prospecting in environments with naturally elevated CO₂ may uncover novel CO₂-metabolizing organisms with unique characteristics., Importance: Cyanobacteria provide a potential avenue for both biomanufacturing and combatting climate change via high-efficiency photosynthetic carbon sequestration. This study identifies novel photosynthetic organisms isolated from a unique geochemical environment and describes their genomes, growth behavior in culture, and biochemical composition. These cyanobacteria appear to make a tractable research model, and cultures are made publicly available alongside information about their culture and maintenance. Application of these organisms to carbon sequestration and/or biomanufacturing is discussed, including unusual, rapid settling characteristics of the strains relevant to scaled culture.

The therapeutic challenges of orthopedic device-related infections and emerging antimicrobial resistance have attracted attention to drug delivery technologies. This study evaluates the preclinical efficacy of local single- and dual-antibiotic therapy against implant-associated osteomyelitis (IAO) using a drug-eluting depot technology, CarboCell, that provides sustained release of high-dose antibiotics and allows for strategic in situ placement in relation to infectious lesions. Clindamycin and gentamicin were formulated in CarboCell compositions. One-stage-revision of tibial Staphylococcus aureus IAO was conducted in 19 pigs. Pigs were treated locally with CarboCell containing either gentamicin alone for 1 week or a co-formulation of gentamicin and clindamycin for 1 or 3 weeks. Bone, soft tissue, and antibiotic depots were collected for microbiology, histology, and HPLC analyses. Supporting in vivo release studies of CarboCell formulations were performed on mice. Both single- and dual-antibiotic CarboCell formulations were developed and capable of eradicating the infectious bacteria in bone and preventing colonization of implants inserted at revision. Eradication in soft tissue was observed in all pigs after 3 weeks and in 6/9 pigs after 1 week of treatment. Neutrophil counts in bone tissue were below the infection cut-off in all pigs receiving the dual-antibiotic therapies, but above in all pigs receiving the single-antibiotic therapy. Histological signs of active bone reorganization and healing were observed at 3 weeks. In conclusion, all CarboCell formulations demonstrated strong therapeutic activity against IAO, eradicating S. aureus in bone tissue and preventing colonization of implants even without the addition of systemic antibiotic therapy., Competing Interests: A.E.H., J.R.H., and T.L.A. are co-inventors on patents covering the CarboCell technology. All other authors declare no conflict of interest.

Background: Bone infections with Staphylococcus aureus are notoriously difficult to treat and have high recurrence rates. Local antibiotic delivery systems hold the potential to achieve high in situ antibiotic concentrations, which are otherwise challenging to achieve via systemic administration. Existing solutions have been shown to confer suboptimal drug release and distribution. Here we present and evaluate an injectable in situ-forming depot system termed CarboCell. The CarboCell technology provides sustained and tuneable release of local high-dose antibiotics., Methods: CarboCell formulations of levofloxacin or clindamycin with or without antimicrobial adjuvants cis-2-decenoic acid or cis-11-methyl-2-dodecenoic acid were tested in experimental rodent and porcine implant-associated osteomyelitis models. In the porcine models, debridement and treatment with CarboCell-formulated antibiotics was carried out without systemic antibiotic administration. The bacterial burden was determined by quantitative bacteriology., Results: CarboCell formulations eliminated S. aureus in infected implant rat models. In the translational implant-associated pig model, surgical debridement and injection of clindamycin-releasing CarboCell formulations resulted in pathogen-free bone tissues and implants in 9 of 12 and full eradication in 5 of 12 pigs., Conclusions: Sustained release of antimicrobial agents mediated by the CarboCell technology demonstrated promising therapeutic efficacy in challenging translational models and may be beneficial in combination with the current standard of care., Competing Interests: Potential conflicts of interest. A. E. H., J. R. H., and T. L. A. are coinventors on patents covering the CarboCell technology. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Diseases caused by S. pneumoniae are the leading cause of child mortality. As antibiotic resistance of S. pneumoniae is rising, vaccination remains the most recommended solution. However, the existing pneumococcal polysaccharides vaccine (Pneumovax ® 23) proved only to induce T-independent immunity, and strict cold chain dependence of the protein conjugate vaccine impedes its promotion in developing countries, where infections are most problematic. Affordable and efficient vaccines against pneumococcus are therefore in high demand. Here, we present an intranasal vaccine Lipo + CPS12F&αGC, containing the capsular polysaccharides of S. pneumoniae 12F and the iNKT agonist α-galactosylceramide in cationic liposomes. In BALB/cJRj mice, the vaccine effectively activates iNKT cells and promotes B cells maturation, stimulates affinity-matured IgA and IgG production in both the respiratory tract and systemic blood, and displays sufficient protection both in vivo and in vitro. The designed vaccine is a promising, cost-effective solution against pneumococcus, which can be expanded to cover more serotypes and pathogens., (© 2024. The Author(s).)

Motivation: Computational analysis of large-scale metagenomics sequencing datasets have proven to be both incredibly valuable for extracting isolate-level taxonomic, and functional insights from complex microbial communities. However, due to an ever-expanding ecosystem of metagenomics-specific methods and file-formats, designing studies which implement seamless and scalable end-to-end workflows, and exploring the massive amounts of output data have become studies unto themselves. One-click bioinformatics pipelines have helped to organize these tools into targeted workflows, but they suffer from general compatibility and maintainability issues., Methods: To address the gap in easily extensible yet robustly distributable metagenomics workflows, we have developed a module-based metagenomics analysis system: "Core Analysis Metagenomics Pipeline" (CAMP), written in Snakemake, a popular workflow management system, along with a standardized module and working directory architecture. Each module can be run independently or conjointly with a series of others to produce the target data format (ex. short-read preprocessing alone, or short-read preprocessing followed by de novo assembly), and outputs aggregated summary statistics reports and semi-guided Jupyter notebook-based visualizations., Results: We have applied CAMP to a set of ten metagenomics samples to demonstrate how a modular analysis system with built-in data visualization at intermediate steps facilitates rich and seamless inter-communication between output data from different analytic purposes., Availability: The module template as well as the modules described below can be found at https://github.com/MetaSUB-CAMP., Competing Interests: Competing Interests No competing interest is declared.

Cancer curing immune responses against heterogeneous solid cancers require that a coordinated immune activation is initiated in the antigen avid but immunosuppressive tumor microenvironment (TME). The plastic TME, and the poor systemic tolerability of immune activating drugs are, however, fundamental barriers to generating curative anticancer immune responses. Here, we introduce the CarboCell technology to overcome these barriers by forming an intratumoral sustained drug release depot that provides high payloads of immune stimulatory drugs selectively within the TME. The CarboCell thereby induces a hot spot for immune cell training and polarization and further drives and maintains the tumor-draining lymph nodes in an anticancer and immune activated state. Mechanistically, this transforms cancerous tissues, consequently generating systemic anticancer immunoreactivity. CarboCell can be injected through standard thin-needle technologies and has inherent imaging contrast which secure accurate intratumoral positioning. In particular, here we report the therapeutic performance for a dual-drug CarboCell providing sustained release of a Toll-like receptor 7/8 agonist and a transforming growth factor-β inhibitor in preclinical tumor models in female mice., (© 2024. The Author(s).)

Objectives: In patients with epithelial ovarian cancer (EOC), the clinical efficacy of monotherapy with immune checkpoint inhibitors (ICIs) against PD-1/PD-L1 is modest. To enhance response rates to these immunotherapeutic agents and broaden the indications for their use, new approaches involving combinational therapy are needed. The immune regulator CD73 is a potential target, as it promotes tumor escape by producing immunosuppressive extracellular adenosine in the tumor microenvironment. Here, we present the results from the NSGO-OV-UMB1/ENGOT-OV-30 trial evaluating the activity of combining the anti-CD73 antibody oleclumab with the anti-PD-L1 checkpoint inhibitor durvalumab in patients with recurrent EOC., Methods: In this phase II open-label non-randomized study, patients with CD73-positive relapsed EOC were intravenously administered oleclumab (3000 mg, Q2W) and durvalumab (1500 mg, Q4W). The primary endpoint was disease control rate (DCR) at 16 weeks. The expression of PD-L1 and CD8 was assessed by immunohistochemistry of archival tumors., Results: This trial included 25 patients with a median age of 66 years (47-77 years). Twenty-two patients were evaluable for treatment activity analysis. The DCR was 27%, the median progression-free survival was 2.7 months (95% CI: 2.2-4.2) and the median overall survival was 8.4 months (95% CI: 5.0-13.4). Infiltration of CD8 + cells and PD-L1 expression on tumor cells were observed in partially overlapping sets of 74% of the tumor samples. Neither CD8- nor PD-L1-positivity were significantly associated with better DCR., Conclusions: Combined treatment with oleclumab and durvalumab was safe and demonstrated limited anti-tumor activity in patients with recurrent EOC., Competing Interests: Declaration of competing interest The authors declare the following financial interest / personal relationship which may be consider as potential competing interests: MRM reports receiving an institutional study grant and investigational medicinal product from AstraZeneca (no personal grants were received). JM reports having received consulting fee payments from GlaxoSmithKline, AstraZeneca and Eisai; receiving payment or honoraria for lectures, presentations, speakers' bureaus, manuscript writing or educational events from Eisai; and participation on a data safety monitoring board or advisory board of Eisai. KL reports receiving research funding from GlaxoSmithKline; receiving consulting fee payments from AstraZeneca, Merck Sharp and Dohme, Nykode, Eisai and GlaxoSmithKline; and being on the safety monitoring board of Karyopharm. AA reports participation on the advisory boards of GlaxoSmithKline and Merck Sharp and Dohme. LAA reports receiving grants or contracts from the Research Council of Norway. LCVT reports receiving personal fees for lectures, presentations, speakers' bureaus, manuscript writing or educational events from Bayer and AstraZeneca; and receiving personal fee payments from Eisai for participating on a data safety monitoring board or Advisory Board. KM reports receiving speakers' honoraria from GlaxoSmithKline and AstraZeneca; receiving compensation for travel expenses from GlaxoSmithKline and AstraZeneca; participating in a trial-specific safety review committee for Kancera AB; and being the deputy medical director for NSGO-CTU. LB reports receiving speakers' honoraria from GlaxoSmithKline, and Merck Sharp and Dohme; having leadership roles in Onkologisk Forum between 2018 and 2022, and in the NSGO and NSGO-CTU since 2021; and receiving investigational medicinal product from AstraZeneca for a researcher-initiated trial. LT, JRH, RdPC, MW, HR and SNL report no personal conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

Liposomes carrying chemotherapeutic drugs can accumulate passively in solid tumors at high levels. However, additional targeting of the liposomes towards e.g. receptors expressed on cancer cells may improve their interaction and therapeutic properties. In this study, we designed a liposomal delivery system, which utilizes the intrinsic characteristics of HER2-positive tumors to ensure efficient delivery of oxaliplatin to the cancer cells. On the liposome surface, trastuzumab, an antibody specific to the HER2 receptor, was shown to facilitate internalization by the cancer cells. A polyethylene glycol (PEG) layer on the liposome surface provides protection from mononuclear phagocyte system uptake. To optimize the interaction between liposomes and cancer cells, a protease-sensitive cleavable peptide linker was inserted at the base of each PEG. The PEG layer is then cleaved off by intra- and extracellular matrix metalloproteinases (MMPs) upon accumulation in the tumor. Our data demonstrate that the removal of PEG significantly destabilizes the liposomes and leads to substantial oxaliplatin release. The proposed beneficial effect of combining antibody-mediated internalization with MMP sensitivity was confirmed in a series of in vivo studies using ovarian cancer xenograft models. The results demonstrated that HER2-targeted MMP-sensitive liposomes have superior anticancer activity compared to non-targeted and non-cleavable liposomes., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023. Published by Elsevier B.V.)

iNKT cells - often referred as the "Swiss Army knife" of the immune system - have emerged as central players in cancer vaccine therapies. Glycolipids activating iNKT cells, such as α-galactosylceramide (αGalCer), can enhance the immune response against co-delivered cancer antigens and have been applied in the design of self-adjuvanting anti-tumor vaccines. In this context, this work focuses on the chemical synthesis of ganglioside tumor-associated carbohydrate antigens (TACAs), namely GM3 and (Neu5Gc)GM3 antigens, their conjugation to αGalCer, and their formulation into liposomes as an efficient platform for their in vivo delivery. Liposomes containing GM3-αGalCer, (Neu5Gc)GM3-αGalCer, and equimolar amounts of the two conjugates have been fully characterized and their ability to activate iNKT cell has been confirmed ex vivo in mouse and human cell assays. The candidates were tested in in vivo immunization studies, demonstrating an ability to induce both T H 1 and T H 2 cytokines leading to the production of all subclasses of IgG antibodies. Notably, the study also demonstrated that serum antibodies raised against the two TACAs, alone and in combination, were cross-reactive. This finding has consequences for future vaccine designs - even if a highly tumor-selective antigen is chosen, the resulting antibody response may be broader than anticipated., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Approximately 1,000 patients are being diagnosed with renal cell carcinoma in Denmark each year, and 20% of these are metastatic at diagnosis. Renal mass biopsies, developing the diagnostic images improve the diagnosis process. Nephron sparing surgery has been the golden standard for the last decade. Robotic/laparoscopic surgery and ablation therapy have shortened the post-hospital stay and led to a faster recovery. Tyrosine kinase inhibitors and immunotherapy has improved overall survival in the last decade. Despite these great advances, more research is needed to achieve further improvement.

Methylated Homeobox A9 circulating tumor DNA (meth-HOXA9) has been suggested as a blood-based biomarker in epithelial ovarian cancer (EOC), although its prognostic significance remains unproven. The aim of the present study was to investigate the prognostic impact of meth-HOXA9 in patients with recurrent EOC. DNA was purified from 4 mL plasma and, following bilsulfite conversion, meth-HOXA9 was analyzed using a methylation-specific droplet digital PCR. Detection of meth-HOXA9 was reported as a percentage of total DNA and as a binary variable (detectable and undetectable). Meth-HOXA9 status and its dynamics during palliative treatment were correlated with overall survival (OS) as the primary endpoint. At baseline, meth-HOXA9 was detected in 65.9% (83/126) of the patients. The median OS was 8.9 and 17.9 months in patients with detectable and undetectable meth-HOXA9 at baseline (hazard ratio: 2.04, p = 0.002), which remained significant in the multivariate analysis. Median OS in patients with an increase in meth-HOXA9 after one treatment cycle was 5.3 months compared to 33 months in patients with undetectable meth-HOXA9 (p < 0.001). Meth-HOXA9 was significantly related to poor survival and may serve as a prognostic marker in patients with recurrent EOC. The longitudinal monitoring of meth-HOXA9 is clinically feasible with the perspective of aiding clinical decision making.

Systemic administration of toll-like receptor (TLR) agonists have demonstrated impressive preclinical results as an anti-cancer therapy due to their potent innate immune-stimulatory properties. The clinical advancement has, however, been hindered by severe adverse effects due to systemic activation of the immune system. Liposomal drug delivery systems may modify biodistribution, cellular uptake, and extend blood circulation, and thus, potentially enable systemic administration of TLR agonists at therapeutic doses. In this study, we investigated potential barriers for the administration of TLR agonists formulated in polyethylene glycosylated (PEGylated) liposomes with regards to liposome formulation, TLR agonist, administration route, administration schedule, biodistribution, blood clearance, and anti-PEG antibodies. We found that administration of TLR agonists formulated in PEGylated liposomes led to high anti-PEG antibody titers, which upon multiple intravenous administrations, resulted in accelerated blood clearance and acute hypersensitivity reactions. The latter was found to be associated with anti-PEG IgG antibody and not anti-PEG IgM antibody opsonization. This study highlights the need to carefully design and evaluate nanoparticle delivery systems for immunotherapy as anti-nanoparticle immune responses may challenge the therapeutic application., (Copyright © 2021. Published by Elsevier B.V.)

The technological leap of DNA sequencing generated a tension between modern metagenomics and historical microbiology. We are forcibly harmonizing the output of a modern tool with centuries of experimental knowledge derived from culture-based microbiology. As a thought experiment, we borrow the notion of Cartesian doubt from philosopher Rene Descartes, who used doubt to build a philosophical framework from his incorrigible statement that "I think therefore I am." We aim to cast away preconceived notions and conceptualize microorganisms through the lens of metagenomic sequencing alone. Specifically, we propose funding and building analysis and engineering methods that neither search for nor rely on the assumption of independent genomes bound by lipid barriers containing discrete functional roles and taxonomies. We propose that a view of microbial communities based in sequencing will engender novel insights into metagenomic structure and may capture functional biology not reflected within the current paradigm.

Adoptive T-cell transfer (ACT) offers a curative therapeutic option for subsets of melanoma and hematological cancer patients. To increase response rates and broaden the applicability of ACT, it is necessary to improve the post-infusion performance of the transferred T cells. The design of improved treatment strategies includes transfer of cells with a less differentiated phenotype. Such T cell subsets have high proliferative potential but require stimulatory signals in vivo to differentiate into tumor-reactive effector T cells. Thus, combination strategies are needed to support the therapeutic implementation of less differentiated T cells. Here we show that systemic delivery of tumor-associated antigens (TAAs) facilitates in vivo priming and expansion of previously non-activated T cells and enhance the cytotoxicity of activated T cells. To achieve this in vivo priming, we use flexible delivery vehicles of TAAs and a TLR7/8 agonist. Contrasting subcutaneous delivery systems, these vehicles accumulate TAAs in the spleen, thereby achieving close proximity to both cross-presenting dendritic cells and transferred T cells, resulting in robust T-cell expansion and anti-tumor reactivity. This TAA delivery platform offers a strategy to safely potentiate the post-infusion performance of T cells using low doses of antigen and TLR7/8 agonist, and thereby enhance the effect of ACT., (© 2021. The Author(s).)

The Virus-X-Viral Metagenomics for Innovation Value-project was a scientific expedition to explore and exploit uncharted territory of genetic diversity in extreme natural environments such as geothermal hot springs and deep-sea ocean ecosystems. Specifically, the project was set to analyse and exploit viral metagenomes with the ultimate goal of developing new gene products with high innovation value for applications in biotechnology, pharmaceutical, medical, and the life science sectors. Viral gene pool analysis is also essential to obtain fundamental insight into ecosystem dynamics and to investigate how viruses influence the evolution of microbes and multicellular organisms. The Virus-X Consortium, established in 2016, included experts from eight European countries. The unique approach based on high throughput bioinformatics technologies combined with structural and functional studies resulted in the development of a biodiscovery pipeline of significant capacity and scale. The activities within the Virus-X consortium cover the entire range from bioprospecting and methods development in bioinformatics to protein production and characterisation, with the final goal of translating our results into new products for the bioeconomy. The significant impact the consortium made in all of these areas was possible due to the successful cooperation between expert teams that worked together to solve a complex scientific problem using state-of-the-art technologies as well as developing novel tools to explore the virosphere, widely considered as the last great frontier of life., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Increasing numbers of lung tumors are identified at early disease stages by diagnostic imaging in screening programs, but difficulties in locating these during surgical intervention has prevented an improved treatment outcome. Surgical biomarkers that are visible on diagnostic images, and that provide the surgeon with real-time image guidance during the intervention are thus highly warranted to bridge diagnostic precision into enhanced therapeutic outcome. In this paper, a liquid soft tissue marker for near infrared fluorescence and radio-guidance is presented. The biocompatible marker is based on the carbohydrate ester, sucrose acetate isobutyrate, ethanol, and a multifunctional naphthalocyanine dye, which enable near infrared fluorescence image-guided resection at short, medium and long tissue depths. Naphthalocyanine dyes have high quantum yields and may further act as chelators of radionuclides. Upon injection of the liquid marker, a gel-like depot is formed in situ at the site of injection, wherein the fluorescent dye and radionuclide is retained. The radiolabeled markers were optimized for minimal fluorescence quenching and high retention of the positron emission tomography radionuclide 64 Cu. The performance of the radiolabeled marker was tested in vivo in mice, where it displayed high photostability over a period of 4 weeks, and high retention of 64 Cu for 48 hours. The retention and biodistribution of 64 Cu was quantified via PET/CT, and the fluorescence emission by an in vivo imaging system. The presented data demonstrate proof-of-concept for naphthalocyanine markers as multimodal imaging agents that can bridge the precision of diagnostic imaging into surgical interventions., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Local application of radioactive sources as brachytherapy is well established in oncology. This treatment is highly invasive however, due to the insertion of millimeter sized metal seeds. The authors report the development of a new concept for brachytherapy, based on gold-palladium (AuPd) alloy nanoparticles, intrinsically radiolabeled with 103 Pd. These are formulated in a carbohydrate-ester based liquid, capable of forming biodegradable gel-like implants upon injection. This allows for less invasive administration through small-gauge needles. [ 103 Pd]AuPd nanoparticles with sizes around 20 nm are prepared with radiolabeling efficiencies ranging from 79% to >99%. Coating with the hydrophobic polymer poly(N-isopropylacrylamide) leads to nanoparticle diameters below 40 nm. Dispersing the nanoparticles in ethanol with water insoluble carbohydrate esters gives "nanogels", a low viscosity liquid capable of solidifying upon injection into aqueous environments. Both nanoparticles and radioactivity are stably retained in the nanogel over 25 days (>99%) after formation in aqueous buffers. Animals bearing CT26 murine tumors are injected intratumorally with 25 MBq of the 103 Pd-nanogel, and display tumor growth delay and significantly increase median survival times compared with control groups. Excellent retention in the tumor of both the 103 Pd and the nanoparticle matrix itself is observed, demonstrating a potential for replacing currently used brachytherapy seeds., (© 2021 Wiley-VCH GmbH.)

The main structural element defining the cell is the lipid membrane, which is an integral part of regulating the fluxes of ion and nutrition molecules in and out of the cell. Surprisingly, copper ions were found to have anomalous membrane permeability. This led us to consider a broader spectrum of cations and further a new approach for using liposomes as nanoreactors for synthesis of metal and metal alloy nanoparticles. In the present study, the high membrane permeability of Cu2+ and its neighbouring transition elements in the periodic table was investigated. The permeability of Ni2+, Cu2+, Zn2+, Ag+, Au3+, Mg2+, Ca2+ and Lu3+ was assessed, and we report that Zn2+, Cu2+, Ag+ and Au3+ surprisingly are able to cross lipid bilayers. This knowledge is highly relevant for understanding trafficking of cations in biological systems, as well as for design of novel nanoparticle and nanoreactor systems. An example of its use is presented as a platform for synthesizing single highly uniform gold nanoparticles inside liposomal nanoreactors. We envision that this approach could provide a new nanoreactor methodology for forming highly structurally constrained uniform metal and metal alloy nanoparticles, as well as new methods for in vivo tracking of liposomes.

Background: The accumulation of liposome encapsulated chemotherapy in solid cancers is dependent on the presence of the enhanced permeability and retention (EPR) effect. Positron emission tomography (PET) imaging with a liposome encapsulated radioisotope, such as liposome encapsulated Cu-64 ( 64 Cu-liposome) may help to identify tumors with high liposome accumulation, and thereby stratify patients based on expected benefit from liposomal chemotherapy. However, intravenous administration of liposomes without a cytotoxic content is complicated by the accelerated blood clearance (ABC) phenomenon for succeeding therapeutic liposome dosing. Alternative markers for assessing the tumor's EPR level are therefore warranted., Materials and Methods: To increase our understanding of EPR variations and to ultimately identify an alternative marker for the EPR effect, we investigated the correlation between 64 Cu-liposome PET/CT (EPR effect) and 68 Ga-RGD PET/CT (neoangiogenesis), 18 F-FDG PET/CT (glycolysis), diffusion-weighted MRI (diffusivity) and interstitial fluid pressure in two experimental cancer models (CT26 and COLO 205)., Results: 64 Cu-liposome and 68 Ga-RGD SUV max displayed a significant moderate correlation, however, none of the other parameters evaluated displayed significant correlations. These results indicate that differences in neoangiogenesis may explain some EPR variability, however, as correlations were only moderate and not observed for SUV mean , 68 Ga-RGD is probably insufficient to serve as a stand-alone surrogate marker for quantifying the EPR effect and stratifying patients., Competing Interests: Financial support was provided by the Danish Strategic Research Council (NABIIT) ref. 2106-07-0033, the Lundbeck Foundation and the European Council (ERC grant). Annemarie Thuri Kristensen reports grants from Danish Technical University, during the conduct of the study; grants from Novo Nordisk A/S, grants from Copenhagen ZOO, grants from Independent Research Fund Denmark, outside the submitted work. The authors report no other potential conflicts of interest in this work., (© 2020 Børresen et al.)

Objective: Recent research indicated favorable prognostic impact of intratumoral natural killer (NK) cells in ovarian carcinoma (OC). The role of NK cells during chemotherapy in OC is unknown. We investigated impact of NK cells in OC patients treated with palliative chemotherapy., Methods: Participants receiving palliative chemotherapy for recurrent OC ( N  = 72) had prospectively blood samples at baseline and before cycle 2. NK cell counts were quantified by flow cytometry. NK cell activity was measured by the NK Vue ® assay, estimating interferon-gamma production. Overall survival (OS) was the primary endpoint. Cutoffs were predefined, NK numbers (≥184 × 10 6 cells/L vs. <184 × 10 6 cells/L) and NK activity (<200 pg/mL vs. ≥200 pg/mL)., Results: Median OS in patients with low vs. high NK cell count at baseline was 7.1 months vs. 15.6 months ( p  = .028), respectively, and before cycle 2 was 5.7 vs. 17.3 months, p  < .001, respectively. The difference in restricted mean survival (ΔRMST) was 5.7 months (95% CI: 3.3-8.0) at cycle 2 vs. 2.5 months (95% CI: -0.6 to 5.6) at baseline, showing a significant difference with no overlap of confidence intervals. In multivariate analyses, low NK cell count remained significant with a hazard ratio (HR)=2.83, 95% CI: 1.53-5.22, p  = .001 (baseline) and HR = 3.34, 95% CI: 1.67-6.71, p  = .001 (before cycle 2). Patients with both low NK count and NK activity at baseline ( N  = 20) had median OS 6.5 months vs. 11.5 months in patients with either high activity, high count or both ( p  = .007). In parallel, patients with both low NK activity and count at cycle 2 ( N  = 18) had a median survival of 4.0 months vs. 15.4 months ( p  < .001)., Conclusions: A low blood NK cell count in recurrent metastatic ovarian cancer during chemotherapy is associated with unfavorable prognostic impact. Early increase in survival difference based on NK cell status suggests an association between NK cell count and treatment benefit.

Immunosuppressive cells in the tumor microenvironment allow cancer cells to escape immune recognition and support cancer progression and dissemination. To improve therapeutic efficacy, we designed a liposomal oxaliplatin formulation (PCL8-U75) that elicits cytotoxic effects toward both cancer and immunosuppressive cells via protease-mediated, intratumoral liposome activation. The PCL8-U75 liposomes displayed superior therapeutic efficacy across all syngeneic cancer models in comparison to free-drug and liposomal controls. The PCL8-U75 depleted myeloid-derived suppressor cells and tumor-associated macrophages in the tumor microenvironment. The combination of improved cancer cell cytotoxicity and depletion of immunosuppressive populations of immune cells is attractive for combination with immune-activating therapy. Combining the PCL8-U75 liposomes with a TLR7 agonist induced immunological rejection of established tumors. This combination therapy increased intratumoral numbers of cancer antigen-specific cytotoxic T cells and Foxp3 - T helper cells. These results are encouraging toward advancing liposomal drug delivery systems with anticancer and immune-modulating properties into clinical cancer therapy., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

Diagnostic imaging often outperforms the surgeon's ability to identify small structures during therapeutic procedures. Smart soft tissue markers that translate the sensitivity of diagnostic imaging into optimal therapeutic intervention are therefore highly warranted. This paper presents a unique adaptable liquid soft tissue marker system based on functionalized carbohydrates (Carbo-gel). The liquid state of these markers allows for high-precision placement under image guidance using thin needles. Based on step-by-step modifications, the image features and mechanical properties of markers can be optimized to bridge diagnostic imaging and specific therapeutic interventions. The performance of Carbo-gel is demonstrated for markers that (i) have radiographic, magnetic resonance, and ultrasound visibility; (ii) are palpable and visible; and (iii) are localizable by near-infrared fluorescence and radio guidance. The study demonstrates encouraging proof of concept for the liquid marker system as a well-tolerated multimodal imaging marker that can improve image-guided radiotherapy and surgical interventions, including robotic surgery., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

Introduction: The aim of the present study was to investigate the prognostic impact of intratumoral cytotoxic T cells, Natural Killer (NK) cells, neutrophils and PD-L1 expression in patients with epithelial ovarian cancer. Methods: All patients diagnosed with high-grade serous carcinoma (HGSC) in Denmark in 2005 were included in the study. Immunohistochemical staining for PD-L1, CD8, CD66b and CD57 was performed on tumor tissue from 283 patients. Cell densities were analyzed using a digital image analysis method. The primary endpoint was overall survival (OS). Results: The median OS for HGSC patients was 30 months. It was 45 months in patients with high level of CD57+ NK cells (≥10 cells/mm 2 ) compared with 29 month in patients with low level (<10 cells/mm 2 ) ( p  = .0310). The median OS was 37 and 25 months in patients with high vs. low level of CD8+ T cells (cutoff 80 cells/mm 2 ) ( p  = .0008). In multivariate analysis, high numbers of CD57+ NK cells and CD8+ T cells remained independent markers of favorable OS, adjusted hazard ratio (HR) 0.67; p  = .041, and HR 0.72; p  = .020, respectively. PD-L1 expression was associated with improved OS (37 months vs. 22 months, p  = .0006), but was only borderline significant in the multivariate analysis (HR 0.77, p  = .061). CD66b + neutrophils had no association with OS. Conclusions: In patients with HGSC tumor-infiltrating CD57+ NK cells and CD8+ T cells had favorable prognostic impact, while PD-L1 expression had borderline favorable prognostic significance. CD66b + neutrophils had no prognostic association. These findings may influence future immunotherapy development.

Purpose: Biomarkers are needed to guide treatment decisions in recurrent ovarian cancer, as a high proportion of patients do not benefit from treatments. Data on immune subsets in patients receiving chemotherapy are scarce. We investigated the impact of T cells, B cells, neutrophils and the neutrophil-lymphocyte ratio (NLR) in ovarian cancer patients receiving palliative chemotherapy., Methods: Blood samples were collected prospectively at baseline in recurrent ovarian cancer (N = 72) receiving chemotherapy. T cells, B cells, neutrophils, and NLR were analyzed. Primary and secondary endpoints were overall survival (OS) and treatment response, respectively. Cut-offs for T and B cells were predefined., Results: In patients with low vs. high T and B cells counts, OS was 6.1 months vs 12.0 months (P = 0.017) and 6.1 months vs 12.0 months (P = 0.011, respectively. Low T and B cells analyzed as continuous variables were also associated with unfavorable OS, P = 0.011 and P = 0.007, respectively. Neutrophils had no significant prognostic impact. Median NLR was 4.1. High vs. low NLR was associated with poor survival, 7.4 months vs. 15.9 months (P = 0.012). In multivariate analysis including platinum sensitivity, number of prior lines of chemotherapy, and performance status, high NLR remained an independent poor prognostic factor HR: 2.17 (95% CI 1.21-3.88) (P = 0.009). High NLR was also significantly associated with lack of response, OR 0.15 (95% CI: 0.04-0.51) (P = 0.002)., Conclusion: In recurrent ovarian cancer patients undergoing palliative chemotherapy, low T and B lymphocyte counts had an unfavorable prognostic impact. High NLR was associated with lack of response and a poor prognosis, and the parameter may be used in patient counselling and treatment decisions.