Generation of Murine Chimeric Antigen Receptor-Modified T Cells for In Vivo Studies in Syngeneic Tumor Models.

CAR-T cell therapy has emerged as a potent and effective tool in the immunotherapy of refractory cancers. However, challenges exist in their clinical application, necessitating extensive preclinical research to optimize their function. Various preclinical in vitro and in vivo models have been proposed for such purpose; among which immunocompetent mouse models serve as an invaluable tool in studying host immune interactions within a more realistic simulation of the tumor milieu. We hereby describe a standardized protocol for the generation of high-titer γ-retroviral vectors through transfection of the HEK293T packaging cell line. The virus-containing supernatant is further concentrated using an inhouse concentrator solution, titrated, and applied to mouse T cells purified via a convenient and rapid method by nylon-wool columns. Using the method presented here, we were able to achieve high titer γ-retrovirus and highly pure mouse T cells with desirable CAR transduction efficiency. The mouse CAR T cells produced through this protocol demonstrate favorable CAR expression and viability, thus making them suitable for further in vitro/in vivo assays. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Production of γ-retroviral vectors from retrovirus-backbone plasmids Basic Protocol 2: Concentration of γ-retrovirus-containing supernatants Basic Protocol 3: Titration of concentrated γ-retrovirus Basic Protocol 4: Isolation and activation of mouse T cells Basic Protocol 5: Transduction of activated mouse T cells, assessment of CAR expression, and expansion of CAR T cells for further in vitro/in vivo studies Support Protocol: Surface staining of cells for flow cytometric assessment of CAR expression.

Ecological and evolutionary dynamics of CRISPR-Cas systems in Clostridium botulinum: Insights from genome mining and comparative analysis.

Understanding the prevalence and distribution of CRISPR-Cas systems across different strains can illuminate the ecological and evolutionary dynamics of Clostridium botulinum populations. In this study, we conducted genome mining to characterize the CRISPR-Cas systems of C. botulinum strains. Our analysis involved retrieving complete genome sequences of these strains and assessing the diversity, prevalence, and evolution of their CRISPR-Cas systems. Subsequently, we performed an analysis of homology in spacer sequences from identified CRISPR arrays to investigate and characterize the range of targeted phages and plasmids. Additionally, we investigated the evolutionary trajectory of C. botulinum strains under selective pressures from foreign invasive DNA. Our findings revealed that 306 strains possessed complete CRISPR-Cas structures, comprising 58% of the studied C. botulinum strains. Secondary structure prediction of consensus repeats indicated that subtype II-C, with longer stems compared to subtypes ID and IB, tended to form more stable RNA secondary structures. Moreover, protospacer motif analysis demonstrated that strains with subtype IB CRISPR-Cas systems exhibited 5'-CGG-3', 5'-CC-3', and 5'-CAT-3' motifs in the 3' flanking regions of protospacers. The diversity observed in CRISPR-Cas systems indicated their classification into subtypes IB, ID, II-C, III-B, and III-D. Furthermore, our results showed that systems with subtype ID and III-D frequently harbored similar spacer patterns. Moreover, analysis of spacer sequences homology with phage and prophage genomes highlighted the specific activities exhibited by subtype IB and III-B against phages and plasmids, providing valuable insights into the functional specialization within these systems.

Simultaneous targeting of Tim3 and A2a receptors modulates MSLN-CAR T cell antitumor function in a human cervical tumor xenograft model.

Introduction: Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of hematological malignancies. However, its efficacy in solid tumors is limited by the immunosuppressive tumor microenvironment that compromises CAR T cell antitumor function in clinical settings. To overcome this challenge, researchers have investigated the potential of inhibiting specific immune checkpoint receptors, including A2aR (Adenosine A2 Receptor) and Tim3 (T cell immunoglobulin and mucin domain-containing protein 3), to enhance CAR T cell function. In this study, we evaluated the impact of genetic targeting of Tim3 and A2a receptors on the antitumor function of human mesothelin-specific CAR T cells (MSLN-CAR) in vitro and in vivo. Methods: Second-generation anti-mesothelin CAR T cells were produced using standard cellular and molecular techniques. A2aR-knockdown and/or Tim3- knockdown anti-mesothelin-CAR T cells were generated using shRNA-mediated gene silencing. The antitumor function of CAR T cells was evaluated by measuring cytokine production, proliferation, and cytotoxicity in vitro through coculture with cervical cancer cells (HeLa cell line). To evaluate in vivo antitumor efficacy of manufactured CAR T cells, tumor growth and mouse survival were monitored in a human cervical cancer xenograft model. Results: In vitro experiments demonstrated that knockdown of A2aR alone or in combination with Tim3 significantly improved CAR T cell proliferation, cytokine production, and cytotoxicity in presence of tumor cells in an antigen-specific manner. Furthermore, in the humanized xenograft model, both double knockdown CAR T cells and control CAR T cells could effectively control tumor growth. However, single knockdown CAR T cells were associated with reduced survival in mice. Conclusion: These findings highlight the potential of concomitant genetic targeting of Tim3 and A2a receptors to augment the efficacy of CAR T cell therapy in solid tumors. Nevertheless, caution should be exercised in light of our observation of decreased survival in mice treated with single knockdown MSLN-CAR T cells, emphasizing the need for careful efficacy considerations.

Exploring the Effectiveness of Ajwain Cream in Treating Taxane-induced Peripheral Neuropathy in Cancer Patients: A Pilot, Randomised and Double-blind Clinical Trial.

Objectives: Chemotherapy-induced peripheral neuropathy is a common disorder among cancer patients receiving various chemotherapeutic protocols. The present study aimed to explore the feasibility of ajwain (Trachyspermum ammi [L.] Sprague) cream in treating peripheral neuropathy symptoms triggered by taxane chemotherapeutic agents. Materials and Methods: This was a pilot, double-blind, and randomised clinical trial on patients with peripheral neuropathy attributable to chemotherapy with taxane drugs during 2021-2022 in Tehran. Patients received ajwain or placebo cream for four weeks and filled out the chemotherapy-induced peripheral neuropathy assessment tool (CIPNAT) at the start and end finale of the trial. Side effects were also noted. Results: Thirty patients suffering from breast, lung, gastro-intestinal, or prostate cancer were allocated to each of the drug and placebo groups. The mean difference in CIPNAT score between the groups was 0.83, demonstrating the statistical ineffectiveness of the drug compared with the placebo (P = 0.372). The safety profile showed promising outcomes at the end of the trial. Conclusion: Although the effectiveness of ajwain cream was unacceptable in treating chemotherapy-induced peripheral neuropathy symptoms, multicentre controlled trials with ample sample size are mandatory for an all-inclusive inference.

PX-478, an HIF-1α inhibitor, impairs mesoCAR T cell antitumor function in cervical cancer.

Introduction: Chimeric Antigen Receptor (CAR) T cell therapy has demonstrated remarkable success in treating hematological malignancies. However, its efficacy against solid tumors, including cervical cancer, remains a challenge. Hypoxia, a common feature of the tumor microenvironment, profoundly impacts CAR T cell function, emphasizing the need to explore strategies targeting hypoxia-inducible factor-1α (HIF-1α). Methods: In this study, we evaluated the effects of the HIF-1α inhibitor PX-478 on mesoCAR T cell function through in-silico and in vitro experiments. We conducted comprehensive analyses of HIF-1α expression in cervical cancer patients and examined the impact of PX-478 on T cell proliferation, cytokine production, cytotoxicity, and exhaustion markers. Results: Our in-silico analyses revealed high expression of HIF-1α in cervical cancer patients, correlating with poor prognosis. PX-478 effectively reduced HIF-1α levels in T and HeLa cells. While PX-478 exhibited dose-dependent inhibition of antigen-nonspecific T and mesoCAR T cell proliferation, it had minimal impact on antigen-specific mesoCAR T cell proliferation. Notably, PX-478 significantly impaired the cytotoxic function of mesoCAR T cells and induced terminally exhausted T cells. Discussion: Our results underscore the significant potential and physiological relevance of the HIF-1α pathway in determining the fate and function of both T and CAR T cells. However, we recognize the imperative for further molecular investigations aimed at unraveling the intricate downstream targets associated with HIF-1α and its influence on antitumor immunity, particularly within the context of hypoxic tumors. These insights serve as a foundation for the careful development of combination therapies tailored to counter immunosuppressive pathways within hypoxic environments and fine-tune CAR T cell performance in the intricate tumor microenvironment.

Retraction notice to "The therapeutic potential of human adipose-derived mesenchymal stem cells producing CXCL10 in a mouse melanoma lung metastasis model" [Cancer Lett. 419 (2018) 30-39].

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor. After publication, the Editors were contacted by a concerned reader regarding alleged image duplication. These allegations are in regard to Fig. 3a being duplicated from a previously published paper in the journal Stem Cells (Stem Cells. 2008 Sep;26 (9):2332-8. doi: 10.1634/stemcells.2008-0084) and Fig. 8a being duplicated from a previously published paper in the journal Molecular Cancer (Mol Cancer 13, 255 (2014). https://doi.org/10.1186/1476-4598-13-255). After a thorough investigation by the editorial team, the Editors determined that there are multiple identical details between Fig. 5A (Cancer Letters) and Fig. 3A (Stem Cells) and the authors did not produce satisfactory evidence that the published images in Cancer Letters were original. Due to this, the Editor does not have confidence in the results and conclusions presented and has made the decision to retract.

Sexual function index adaptation for breast cancer patients (FSFI-BC)- translation and psychometric properties of Persian version.

BACKGROUND: Effective interventions to improve sexual dysfunction in breast cancer survivors need screening of these dysfunctions with a suitable instrument. The aim of present study was translation and identifying psychometric properties of Female Sexual Function Index - Adapted for Breast Cancer (FSFI-BC) which has been specifically developed for breast cancer survivors. METHOD: This methodological study was performed between February 2017 and October 2018. 200 breast cancer survivors in stage 1 or 2 who were selected through convenience sampling method, completed the questionnaire. Reliability was assessed by Cronbach's alfa and test re-test analysis and construct validity was performed through confirmatory (CFA) and exploratory factor analysis( EFA). RESULTS: Six factors were extracted in exploratory factor analysis (EFA). These factors explained 74.6% of the total variance in in NSA group and 0.821 in SA group. Reliability evaluation indicated high internal consistency and good test re-test reliability. Cronbach's alpha coefficient in all areas of the tool was above 0.7 (the lowest and the highest measures were 0.885 and 0.945, respectively), which is a good indicator for reliability of an instrument. Confirmatory factor analysis showed an acceptable fitness for seven factors of FSFI-BC questionnaire (Normed Fit Index or NFI = 0.9 for both groups, Comparative of Fit Index or CFI = 0.93 and 0.92, χ 2/df = 1.68 and 1.71 for SA(Sexually Active) and NSA(No Sexually Active) individuals, respectively) . CONCLUSION: Study findings suggest that Persian version of FSFI-BC is a suitable instrument for sexual dysfunction screening in breast cancer survivors.

Effect of Honey-Lime Spray as Add-On Therapy on Chemotherapy-Induced Xerostomia in Breast Cancer Patients: A Pilot Randomized Double-Blinded Controlled Trial.

INTRODUCTION: Xerostomia (dry mouth) is a common side effect among patients with cancer undergoing chemotherapy. There is no standard treatment for this symptom yet, although Persian medicine textbooks suggested some products to relieve xerostomia. We aimed to assess the efficacy of honey-lime spray in treating chemotherapy-induced xerostomia in breast cancer patients through a controlled study. METHODS: In this pilot, randomized, double-blinded clinical trial conducted in Shohadaye Tajrish Hospital, Iran, the intervention group received honey-lime spray and nystatin, while the control group used distilled water plus nystatin for 2 weeks. The six-item dry mouth form and visual analog score (VAS) were used to evaluate xerostomia extent and pain, respectively. RESULTS: The standardized value of the difference between the mean scores before and after the study was -10.21 (p < 0.001), and the effect size was estimated at 55%. Also, VAS showed a significant decrease in pain for the intervention group compared with the control group (p < 0.001). There were no serious side effects. CONCLUSION: Honey-lime spray may be a good treatment choice for xerostomia in chemotherapy-induced breast cancer patients, but robust trials with larger samples and prolonged follow-ups are highly recommended.Mundtrockenheit (Xerostomie) ist eine häufige Nebenwirkung bei Krebspatienten, die sich einer Chemotherapie unterziehen. Es gibt bisher keine Standardtherapie für dieses Symptom, doch Lehrbücher der persischen Medizin schlagen einige Mittel zur Linderung von Mundtrockenheit vor. Unser Ziel war es, die Wirksamkeit von Honig-Limetten-Spray zur Behandlung von chemotherapiebedingter Mundtrockenheit bei Patientinnen mit Brustkrebs im Rahmen einer kontrollierten Studie zu untersuchen.In dieser randomisierten, doppelt verblindeten klinischen Pilotstudie, die im Shohadaye Tajrish Hospital im Iran durchgeführt wurde, erhielt die Interventionsgruppe zwei Wochen lang Honig-Limetten-Spray und Nystatin und die Kontrollgruppe destilliertes Wasser plus Nystatin. Ein Mundtrockenheits-Fragebogen mit sechs Items wurde zur Beurteilung der Schwere der Mundtrockenheit verwendet und eine visuelle Analogskala zur Beurteilung der Schmerzen.Der standardisierte Wert der Differenz zwischen den mittleren Scores vor und nach der Studie betrug −10,21 (p < 0,01), und die geschätzte Effektstärke betrug 55%. Zudem zeigte die VAS eine signifikante Abnahme der Schmerzen in der Interventionsgruppe im Vergleich zur Kontrollgruppe (p < 0,01). Es traten keine schwerwiegenden Nebenwirkungen auf.Honig-Limetten-Spray könnte eine gute Wahl für die Behandlung von Mundtrockenheit bei Brustkrebspatientinnen unter Chemotherapie sein, doch hierzu werden belastbare Studien mit größerem Probenumfang und längerer Nachbeobachtung nachdrücklich empfohlen.

Applications of single-cell omics for chimeric antigen receptor T cell therapy.

Chimeric antigen receptor (CAR) T cell therapy is a promising cancer treatment modality. The breakthroughs in CAR T cell therapy were, in part, possible with the help of cell analysis methods, such as single-cell analysis. Bulk analyses have provided invaluable information regarding the complex molecular dynamics of CAR T cells, but their results are an average of thousands of signals in CAR T or tumour cells. Since cancer is a heterogeneous disease where each minute detail of a subclone could change the outcome of the treatment, single-cell analysis could prove to be a powerful instrument in deciphering the secrets of tumour microenvironment for cancer immunotherapy. With the recent studies in all aspects of adoptive cell therapy making use of single-cell analysis, a comprehensive review of the recent preclinical and clinical findings in CAR T cell therapy was needed. Here, we categorized and summarized the key points of the studies in which single-cell analysis provided insights into the genomics, epigenomics, transcriptomics and proteomics as well as their respective multi-omics of CAR T cell therapy.

Outsmarting trogocytosis to boost CAR NK/T cell therapy.

Chimeric antigen receptor (CAR) NK and T cell therapy are promising immunotherapeutic approaches for the treatment of cancer. However, the efficacy of CAR NK/T cell therapy is often hindered by various factors, including the phenomenon of trogocytosis, which involves the bidirectional exchange of membrane fragments between cells. In this review, we explore the role of trogocytosis in CAR NK/T cell therapy and highlight potential strategies for its modulation to improve therapeutic efficacy. We provide an in-depth analysis of trogocytosis as it relates to the fate and function of NK and T cells, focusing on its effects on cell activation, cytotoxicity, and antigen presentation. We discuss how trogocytosis can mediate transient antigen loss on cancer cells, thereby negatively affecting the effector function of CAR NK/T cells. Additionally, we address the phenomenon of fratricide and trogocytosis-associated exhaustion, which can limit the persistence and effectiveness of CAR-expressing cells. Furthermore, we explore how trogocytosis can impact CAR NK/T cell functionality, including the acquisition of target molecules and the modulation of signaling pathways. To overcome the negative effects of trogocytosis on cellular immunotherapy, we propose innovative approaches to modulate trogocytosis and augment CAR NK/T cell therapy. These strategies encompass targeting trogocytosis-related molecules, engineering CAR NK/T cells to resist trogocytosis-induced exhaustion and leveraging trogocytosis to enhance the function of CAR-expressing cells. By overcoming the limitations imposed by trogocytosis, it may be possible to unleash the full potential of CAR NK/T therapy against cancer. The knowledge and strategies presented in this review will guide future research and development, leading to improved therapeutic outcomes in the field of immunotherapy.