MHC class I and II-deficient humanized mice are suitable tools to test the long-term antitumor efficacy of immune checkpoint inhibitors and T-cell engagers.

BACKGROUND: Immunodeficient mice engrafted with peripheral blood mononuclear cells (PBMCs) are models to study new cancer immunotherapy agents. However, this approach is associated with xenograft-versus-host disease (xGVHD), which starts early after PBMC transfer and limits the duration and interpretation of experiments. Here, we explore different approaches to overcome xGVHD and better support the development of cancer immunotherapies. METHODS: Immunodeficient NOD-scid IL2Rgnull (NSG) mice were intravenously transferred with human PBMCs and subcutaneously co-engrafted with HT29 human colon carcinoma cells. Diverse strategies to reduce xGVHD while preserving the antitumor activity of human immune cells were evaluated: (1) ex vivo immune graft modification by depleting CD4+ T cells pre-transfer using magnetic beads, (2) post-transplantation cyclophosphamide administration to eliminate proliferating xenoreactive T-cell clones and (3) using major histocompatibility complex (MHC) class I and II-deficient NSG mice: (Kb Db)null (IA)null (MHC-dKO NSG). Body weight and plasma murine alanine aminotransferase levels were measured as indicators of xGVHD and tumor size was measured every 2-3 days to monitor antitumor activity. The antitumor effects and pharmacodynamics of nivolumab plus ipilimumab and an anti-epithelial cell adhesion molecule (EpCAM)/CD3 T-cell engager (αEpCAM/CD3 bispecific antibody (BsAb)) were evaluated in the model. RESULTS: CD4+ T-cell depletion attenuates xGVHD but also abrogates the antitumor activity. Cyclophosphamide limits the antitumor response and does not substantially prevent xGVHD. In contrast, xGVHD was significantly attenuated in MHC-dKO NSG recipients, while the antitumor effect of human PBMCs was preserved. Furthermore, the administration of nivolumab plus ipilimumab caused exacerbated xGVHD in conventional NSG mice, thereby precluding the observation of their antitumor effects. Severe xGVHD did not occur in MHC-dKO NSG mice thus enabling the study of complete and durable tumor rejections. Similarly, NSG mice treated with an αEpCAM/CD3 BsAb showed complete tumor regressions, but died due to xGVHD. In contrast, MHC-dKO NSG mice on treatment with the αEpCAM/CD3 BsAb achieved complete tumor responses without severe xGVHD. A significant proportion of mice rendered tumor-free showed tumor rejection on rechallenge with HT29 cells without further treatment. Finally, tumor-infiltrating CD8+ T-cell number increase, activation and CD137 upregulation were observed on αEpCAM/CD3 BsAb treatment. CONCLUSION: Humanized MHC-dKO immunodeficient mice allow and refine the preclinical testing of immunotherapy agents for which experimentation is precluded in conventional immunodeficient mice due to severe xGVHD.

Dietary-Based Diabetes Risk Score and breast cancer: a prospective evaluation in the SUN project.

An association between type 2 diabetes (T2D) and breast cancer risk has been reported. This association can be potentially explained by alteration of the insulin/IGF system. Therefore, we aimed to prospectively investigate whether a previously reported Dietary-Based Diabetes Risk Score (DDS) inversely associated with T2D was also associated with breast cancer risk in the SUN ("Seguimiento Universidad de Navarra") cohort. We followed up 10,810 women (mean age = 35 years, SD = 11 years) for an average of 12.5 years during which 147 new cases of invasive breast cancer were diagnosed. A validated 136-item FFQ was administered at baseline and after 10 years of follow-up. The DDS (range: 11 to 55 points) positively weighted vegetables, fruit, whole cereals, nuts, coffee, low-fat dairy, fiber, PUFA; while it negatively weighted red meat, processed meats, and sugar-sweetened beverages. The DDS was categorized into tertiles. Self-reported medically diagnosed breast cancer cases were confirmed through medical records. We found a significant inverse association between the intermediate tertile of the DDS score and overall breast cancer risk (Hazard ratio, HRT2 vs. T1= 0.55; 95% CI: 0.36-0.82) and premenopausal breast cancer risk (HRT2= 0.26; 95% CI: 0.13-0.53), but not for the highest tertile. This association was stronger among women with a BMI < 25 kg/m2 (pinteraction: 0.029). In conclusion, moderate adherence to the DDS score was associated with a lower risk of breast cancer, especially among premenopausal women and women with a lower BMI. These findings underscore the importance of antidiabetic diet in reducing the risk of breast cancer.

NKG2C/KLRC2 tumor cell expression enhances immunotherapeutic efficacy against glioblastoma.

BACKGROUND: Activating and inhibitory receptors of natural killer (NK) cells such as NKp, NKG2, or CLEC are highly relevant to cold tumors including glioblastoma (GBM). Here, we aimed to characterize the expression of these receptors in GBM to gain insight into their potential role as modulators of the intratumoral microenvironment. METHODS: We performed a transcriptomic analysis of several NK receptors with a focus on the activating receptor encoded by KLRC2, NKG2C, among bulk and single-cell RNA sequencing GBM data sets. We also evaluated the effects of KLRC2-overexpressing GL261 cells in mice treated with or without programmed cell death protein-1 (PD-1) monoclonal antibody (mAb). Finally, we analyzed samples from two clinical trials evaluating PD-1 mAb effects in patients with GBM to determine the potential of NKG2C to serve as a biomarker of response. RESULTS: We observed significant expression of several inhibitory NK receptors on GBM-infiltrating NK and T cells, which contrasts with the strong expression of KLRC2 on tumor cells, mainly at the infiltrative margin. Neoplastic KLRC2 expression was associated with a reduction in the number of myeloid-derived suppressor cells and with a higher level of tumor-resident lymphocytes. A stronger antitumor activity after PD-1 mAb treatment was observed in NKG2Chigh-expressing tumors both in mouse models and patients with GBM whereas the expression of inhibitory NK receptors showed an inverse association. CONCLUSIONS: This study explored the role of neoplastic NKG2C/KLRC2 expression in shaping the immune profile of GBM and suggests that it is a predictive biomarker for positive responses to immune checkpoint inhibitor treatment in patients with GBM. Future studies could further validate this finding in prospective trials.

Spatially resolved tissue imaging to analyze the tumor immune microenvironment: beyond cell-type densities.

INTRODUCTION: The tissue immune microenvironment is associated with key aspects of tumor biology. The interaction between the immune system and cancer cells has predictive and prognostic potential across different tumor types. Spatially resolved tissue-based technologies allowed researchers to simultaneously quantify different immune populations in tumor samples. However, bare quantification fails to harness the spatial nature of tissue-based technologies. Tumor-immune interactions are associated with specific spatial patterns that can be measured. In recent years, several computational tools have been developed to increase our understanding of these spatial patterns. TOPICS COVERED: In this review, we cover standard techniques as well as new advances in the field of spatial analysis of the immune microenvironment. We focused on marker quantification, spatial intratumor heterogeneity analysis, cell‒cell spatial interaction studies and neighborhood analyses.

Low-Dose Ionizing γ-Radiation Elicits the Extrusion of Neutrophil Extracellular Traps.

PURPOSE: Patients with cancer frequently undergo radiotherapy in their clinical management with unintended irradiation of blood vessels and copiously irrigated organs in which polymorphonuclear leukocytes circulate. Following the observation that such low doses of ionizing radiation are able to induce neutrophils to extrude neutrophil extracellular traps (NET), we have investigated the mechanisms, consequences, and occurrence of such phenomena in patients undergoing radiotherapy. EXPERIMENTAL DESIGN: NETosis was analyzed in cultures of neutrophils isolated from healthy donors, patients with cancer, and cancer-bearing mice under confocal microscopy. Cocultures of radiation-induced NETs, immune effector lymphocytes, and tumor cells were used to study the effects of irradiation-induced NETs on immune cytotoxicity. Radiation-induced NETs were intravenously injected to mice for assessing their effects on metastasis. Circulating NETs in irradiated patients with cancer were measured using ELISA methods for detecting MPO-DNA complexes and citrullinated histone 3. RESULTS: Irradiation of neutrophils with very low γ-radiation doses (0.5-1 Gy) elicits NET formation in a manner dependent on oxidative stress, NADPH oxidase activity, and autocrine IL8. Radiation-induced NETs interfere with NK cell and T-cell cytotoxicity. As a consequence, preinjection of irradiation-induced NETs increases the number of successful metastases in mouse tumor models. Increases in circulating NETs were readily detected in two prospective series of patients following the first fraction of their radiotherapy courses. CONCLUSIONS: NETosis is induced by low-dose ionizing irradiation in a neutrophil-intrinsic fashion, and radiation-induced NETs are able to interfere with immune-mediated cytotoxicity. Radiation-induced NETs foster metastasis in mouse models and can be detected in the circulation of patients undergoing conventional radiotherapy treatments. See related commentary by Mowery and Luke, p. 3965.

Analysis of Tumor Microenvironment Changes after Neoadjuvant Chemotherapy with or without Bevacizumab in Advanced Ovarian Cancer (GEICO-89T/MINOVA Study).

PURPOSE: The aim of our study was to elucidate the impact of bevacizumab added to neoadjuvant chemotherapy (NACT) on the tumor immune microenvironment and correlate the changes with the clinical outcome of the patients. EXPERIMENTAL DESIGN: IHC and multiplex immunofluorescence for lymphoid and myeloid lineage markers were performed in matched tumor samples from 23 patients with ovarian cancer enrolled in GEICO 1205/NOVA clinical study before NACT and at the time of interval cytoreductive surgery. RESULTS: Our results showed that the addition of bevacizumab to NACT plays a role mainly on lymphoid populations at the stromal compartment, detecting a significant decrease of CD4+ T cells, an increase of CD8+ T cells, and an upregulation in effector/regulatory cell ratio (CD8+/CD4+FOXP3+). None of the changes observed were detected in the intra-epithelial site in any arm (NACT or NACT-bevacizumab). No differences were found in myeloid lineage (macrophage-like). The percentage of Treg populations and effector/regulatory cell ratio in the stroma were the only two variables significantly associated with progression-free survival (PFS). CONCLUSIONS: The addition of bevacizumab to NACT did not have an impact on PFS in the GEICO 1205 study. However, at the cellular level, changes in CD4+, CD8+ lymphocyte populations, and CD8+/CD4+FOXP3 ratio have been detected only at the stromal site. On the basis of our results, we hypothesize about the existence of mechanisms of resistance that could prevent the trafficking of T-effector cells into the epithelial component of the tumor as a potential explanation for the lack of efficacy of ICI in the first-line treatment of advanced epithelial ovarian cancer. See related commentary by Soberanis Pina and Oza, p. 12.

Oncolytic DNX-2401 virotherapy plus pembrolizumab in recurrent glioblastoma: a phase 1/2 trial.

Immune-mediated anti-tumoral responses, elicited by oncolytic viruses and augmented with checkpoint inhibition, may be an effective treatment approach for glioblastoma. Here in this multicenter phase 1/2 study we evaluated the combination of intratumoral delivery of oncolytic virus DNX-2401 followed by intravenous anti-PD-1 antibody pembrolizumab in recurrent glioblastoma, first in a dose-escalation and then in a dose-expansion phase, in 49 patients. The primary endpoints were overall safety and objective response rate. The primary safety endpoint was met, whereas the primary efficacy endpoint was not met. There were no dose-limiting toxicities, and full dose combined treatment was well tolerated. The objective response rate was 10.4% (90% confidence interval (CI) 4.2-20.7%), which was not statistically greater than the prespecified control rate of 5%. The secondary endpoint of overall survival at 12 months was 52.7% (95% CI 40.1-69.2%), which was statistically greater than the prespecified control rate of 20%. Median overall survival was 12.5 months (10.7-13.5 months). Objective responses led to longer survival (hazard ratio 0.20, 95% CI 0.05-0.87). A total of 56.2% (95% CI 41.1-70.5%) of patients had a clinical benefit defined as stable disease or better. Three patients completed treatment with durable responses and remain alive at 45, 48 and 60 months. Exploratory mutational, gene-expression and immunophenotypic analyses revealed that the balance between immune cell infiltration and expression of checkpoint inhibitors may potentially inform on response to treatment and mechanisms of resistance. Overall, the combination of intratumoral DNX-2401 followed by pembrolizumab was safe with notable survival benefit in select patients (ClinicalTrials.gov registration: NCT02798406).

Synplex: In Silico Modeling of the Tumor Microenvironment From Multiplex Images.

Multiplex immunofluorescence is a novel, high-content imaging technique that allows simultaneous in situ labeling of multiple tissue antigens. This technique is of growing relevance in the study of the tumor microenvironment, and the discovery of biomarkers of disease progression or response to immune-based therapies. Given the number of markers and the potential complexity of the spatial interactions involved, the analysis of these images requires the use of machine learning tools that rely for their training on the availability of large image datasets, extremely laborious to annotate. We present Synplex, a computer simulator of multiplexed immunofluorescence images from user-defined parameters: i. cell phenotypes, defined by the level of expression of markers and morphological parameters; ii. cellular neighborhoods based on the spatial association of cell phenotypes; and iii. interactions between cellular neighborhoods. We validate Synplex by generating synthetic tissues that accurately simulate real cancer cohorts with underlying differences in the composition of their tumor microenvironment and show proof-of-principle examples of how Synplex could be used for data augmentation when training machine learning models, and for the in silico selection of clinically relevant biomarkers. Synplex is publicly available at https://github.com/djimenezsanchez/Synplex.

Synergistic effects of combined immunotherapy strategies in a model of multifocal hepatocellular carcinoma.

Immune checkpoint-inhibitor combinations are the best therapeutic option for advanced hepatocellular carcinoma (HCC) patients, but improvements in efficacy are needed to improve response rates. We develop a multifocal HCC model to test immunotherapies by introducing c-myc using hydrodynamic gene transfer along with CRISPR-Cas9-mediated disruption of p53 in mouse hepatocytes. Additionally, induced co-expression of luciferase, EGFP, and the melanosomal antigen gp100 facilitates studies on the underlying immunological mechanisms. We show that treatment of the mice with a combination of anti-CTLA-4 + anti-PD1 mAbs results in partial clearance of the tumor with an improvement in survival. However, the addition of either recombinant IL-2 or an anti-CD137 mAb markedly improves both outcomes in these mice. Combining tumor-specific adoptive T cell therapy to the aCTLA-4/aPD1/rIL2 or aCTLA-4/aPD1/aCD137 regimens enhances efficacy in a synergistic manner. As shown by multiplex tissue immunofluorescence and intravital microscopy, combined immunotherapy treatments enhance T cell infiltration and the intratumoral performance of T lymphocytes.

Weakly supervised deep learning to predict recurrence in low-grade endometrial cancer from multiplexed immunofluorescence images.

Predicting recurrence in low-grade, early-stage endometrial cancer (EC) is both challenging and clinically relevant. We present a weakly-supervised deep learning framework, NaroNet, that can learn, without manual expert annotation, the complex tumor-immune interrelations at three levels: local phenotypes, cellular neighborhoods, and tissue areas. It uses multiplexed immunofluorescence for the simultaneous visualization and quantification of CD68 + macrophages, CD8 + T cells, FOXP3 + regulatory T cells, PD-L1/PD-1 protein expression, and tumor cells. We used 489 tumor cores from 250 patients to train a multilevel deep-learning model to predict tumor recurrence. Using a tenfold cross-validation strategy, our model achieved an area under the curve of 0.90 with a 95% confidence interval of 0.83-0.95. Our model predictions resulted in concordance for 96,8% of cases (κ = 0.88). This method could accurately assess the risk of recurrence in EC, outperforming current prognostic factors, including molecular subtyping.

Characterization of the immune infiltrate in mouse tissue by multiplex immunofluorescence.

Multiplexed immunofluorescence imaging of formalin-fixed, paraffin-embedded (FFPE) specimens mounted on glass slides allow the identification of multiple cell phenotypes while retaining spatial and morphological context. Multiplex immunofluorescence protocols have already been developed and validated for mouse tissues. Immunophenotyping analysis reliably depicts the immune landscape of cancer tissues that has been demonstrated to influence cancer development and progression as well as to have an impact on therapy responsiveness and resistance. Here, we describe a method for multiplexed fluorescence image analysis, enabling analysis of mouse cancer morphology and cell phenotypes in FFPE sections.

Intratumoral Gene Transfer of mRNAs Encoding IL12 in Combination with Decoy-Resistant IL18 Improves Local and Systemic Antitumor Immunity.

IL12-based local gene therapy of cancer constitutes an active area of clinical research using plasmids, mRNAs, and viral vectors. To improve antitumor effects, we have experimentally tested the combination of mRNA constructs encoding IL12 and IL18. Moreover, we have used a form of IL18 [decoy-resistant IL18 (DR-18)] which has preserved bioactivity but does not bind to the IL18 binding protein decoy receptor. Both cytokines dramatically synergize to induce IFNγ release from mouse splenocytes, and, if systemically cotransferred to the liver, they mediate lethal toxicity. However, if given intratumorally to B16OVA tumor-bearing mice, the combination attains efficacy against the directly treated tumor and moderate tumor-delaying activity on distant noninjected lesions. Cotreatment was conducive to the presence of more activated CD8+ T cells in the treated and noninjected tumors. In keeping with these findings, the efficacy of treatment was contingent on the integrity of CD8+ T cells and cDC1 dendritic cells in the treated mice. Furthermore, efficacy of IL12 plus DR-18 local mRNA coinjection against distant concomitant tumors could be enhanced upon combination with anti-PD-1 mAb systemic treatment, thus defining a feasible synergistic immunotherapy strategy.

Reference standards for gene fusion molecular assays on cytological samples: an international validation study.

AIMS: Gene fusions assays are key for personalised treatments of advanced human cancers. Their implementation on cytological material requires a preliminary validation that may make use of cell line slides mimicking cytological samples. In this international multi-institutional study, gene fusion reference standards were developed and validated. METHODS: Cell lines harbouring EML4(13)-ALK(20) and SLC34A2(4)-ROS1(32) gene fusions were adopted to prepare reference standards. Eight laboratories (five adopting amplicon-based and three hybridisation-based platforms) received, at different dilution points two sets of slides (slide A 50.0%, slide B 25.0%, slide C 12.5% and slide D wild type) stained by Papanicolaou (Pap) and May Grunwald Giemsa (MGG). Analysis was carried out on a total of 64 slides. RESULTS: Four (50.0%) out of eight laboratories reported results on all slides and dilution points. While 12 (37.5%) out of 32 MGG slides were inadequate, 27 (84.4%) out of 32 Pap slides produced libraries adequate for variant calling. The laboratories using hybridisation-based platforms showed the highest rate of inadequate results (13/24 slides, 54.2%). Conversely, only 10.0% (4/40 slides) of inadequate results were reported by laboratories adopting amplicon-based platforms. CONCLUSIONS: Reference standards in cytological format yield better results when Pap staining and processed by amplicon-based assays. Further investigation is required to optimise these standards for MGG stained cells and for hybridisation-based approaches.

Cell-Specific Dysregulation of Iron and Oxygen Homeostasis as a Novel Pathophysiology in PSP.

OBJECTIVE: Progressive supranuclear palsy (PSP) is a 4R-tauopathy showing heterogeneous tau cytopathology commencing in the globus pallidus (GP) and the substantia nigra (SN), regions also associated with age-related iron accumulation. Abnormal iron levels have been extensively associated with tau pathology in neurodegenerative brains, however, its role in PSP pathogenesis remains yet unknown. We perform the first cell type-specific evaluation of PSP iron homeostasis and the closely related oxygen homeostasis, in relation to tau pathology in human postmortem PSP brains. METHODS: In brain regions vulnerable to PSP pathology (GP, SN, and putamen), we visualized iron deposition in tau-affected and unaffected neurons, astroglia, oligodendrocytes, and microglia, using a combination of iron staining with immunolabelling. To further explore molecular pathways underlying our observations, we examined the expression of key iron and oxygen homeostasis mRNA transcripts and proteins. RESULTS: We found astrocytes as the major cell type accumulating iron in the early affected regions of PSP, highly associated with cellular tau pathology. The same regions are affected by dysregulated expression of alpha and beta hemoglobin and neuroglobin showing contrasting patterns. We discovered changes in iron and oxygen homeostasis-related gene expression associated with aging of the brain, and identified dysregulated expression of rare neurodegeneration with brain iron accumulation (NBIA) genes associated with tau pathology to distinguish PSP from the healthy aging brain. INTERPRETATION: We present novel aspects of PSP pathophysiology highlighting an overlap with NBIA pathways. Our findings reveal potential novel targets for therapy development and have implications beyond PSP for other iron-associated neurodegenerative diseases. ANN NEUROL 2023;93:431-445.

Biomarkers of tumor-reactive CD4+ and CD8+ TILs associate with improved prognosis in endometrial cancer.

BACKGROUND: Despite the growing interest in immunotherapeutic interventions for endometrial cancer (EC), the prevalence, phenotype, specificity and prognostic value of tumor infiltrating lymphocytes (TILs) in this tumor type remains unclear. METHODS: To better understand the role of TILs in EC, we analyzed the phenotypic traits of CD8+ and CD4+ EC-resident T cells from 47 primary tumors by high-dimensional flow cytometry. In addition, CD8+ and CD4+ TIL subpopulations were isolated based on the differential expression of programmed cell death protein-1 (PD-1) (negative, dim and high) and CD39 (positive or negative) by fluorescence activated cell sorting (FACS), expanded in vitro, and screened for autologous tumor recognition. We further investigated whether phenotypic markers preferentially expressed on CD8+ and CD4+ tumor-reactive TIL subsets were associated with the four distinct molecular subtypes of EC, tumor mutational burden and patient survival. RESULTS: We found that CD8+TILs expressing high levels of PD-1 (PD-1hi) co-expressed CD39, TIM-3, HLA-DR and CXCL13, as compared with TILs lacking or displaying intermediate levels of PD-1 expression (PD-1- and PD-1dim, respectively). Autologous tumor reactivity of sorted and in vitro expanded CD8+ TILs demonstrated that the CD8+PD-1dimCD39+ and PD-1hiCD39+ T cell subsets both contained tumor-reactive TILs and that a higher level of PD-1 expression was associated with increased CD39 and a superior frequency of tumor reactivity. With respect to CD4+ T conventional (Tconv) TILs, co-expression of inhibitory and activation markers was more apparent on PD-1hi compared with PD-1- or PD-1dim T cells, and in fact, it was the CD4+PD-1hi subpopulation that accumulated the antitumor T cells irrespective of CD39 expression. Most importantly, detection of CD8+PD-1hiCD39+ and CD4+PD-1hi tumor-reactive T-cell subsets, but also markers specifically expressed by these subpopulations of TILs, that is, PD-1hi, CD39, CXCL13 and CD103 by CD8+ TILs and PD-1hi and CXCL13 by CD4+ Tconv TILs, correlated with prolonged survival of patients with EC. CONCLUSIONS: Our results demonstrate that EC are frequently infiltrated by tumor-reactive TILs, and that expression of PD-1hi and CD39 or PD-1hi can be used to select and expand CD8+ and CD4+ tumor-reactive TILs, respectively. In addition, biomarkers preferentially expressed on tumor-reactive TILs, rather than the frequency of CD3+, CD8+ and CD4+ lymphocytes, hold prognostic value suggesting their protective role in antitumor immunity.

The association between the tumor immune microenvironments and clinical outcome in low-grade, early-stage endometrial cancer patients.

Endometrial tumors show substantial heterogeneity in their immune microenvironment. This heterogeneity could be used to improve the accuracy of current outcome prediction tools. We assessed the immune microenvironment of 235 patients diagnosed with low-grade, early-stage endometrial cancer. Multiplex quantitative immunofluorescence was carried out to measure CD8, CD68, FOXP3, PD-1, and PD-L1 markers, as well as cytokeratin (CK), on tissue microarrays. Clustering results revealed five robust immune response patterns, each associated with specific immune populations, cell phenotypes, and cell spatial clustering. Most samples (69%) belonged to the immune-desert subtype, characterized by low immune cell densities. Tumor-infiltrating lymphocyte (TIL)-rich samples (4%) displayed high CD8+ T-cell infiltration, as well as a high percentage of CD8/PD-1+ cells. Immune-exclusion samples (19%) displayed the lowest CD8+ infiltration combined with high PD-L1 expression levels in CK+ tumor cells. In addition, they demonstrated high tumor cell spatial clustering as well as increased spatial proximity of CD8+ /PD-1+ and CK/PD-L1+ cells. FOXP3 and macrophage-rich phenotypes (3% and 4% of total samples) displayed relatively high levels of FOXP3+ regulatory T-cells and CD68+ macrophages, respectively. These phenotypes correlated with clinical outcomes, with immune-exclusion tumors showing an association with tumor relapse. When compared with prediction models built using routine pathological variables, models optimized with immune variables showed increased outcome prediction capacity (AUC = 0.89 versus 0.78) and stratification potential. The improved prediction capacity was independent of mismatch repair protein status and adjuvant radiotherapy treatment. Further, immunofluorescence results could be partially recapitulated using single-marker immunohistochemistry (IHC) performed on whole tissue sections. TIL-rich tumors demonstrated increased CD8+ T-cells by IHC, while immune-exclusion tumors displayed a lack of CD8+ T-cells and frequent expression of PD-L1 in tumor cells. Our results demonstrate the capability of the immune microenvironment to improve standard prediction tools in low-grade, early-stage endometrial carcinomas. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Targeting the extra domain A of fibronectin for cancer therapy with CAR-T cells.

BACKGROUND: One of the main difficulties of adoptive cell therapies with chimeric antigen receptor (CAR)-T cells in solid tumors is the identification of specific target antigens. The tumor microenvironment can present suitable antigens for CAR design, even though they are not expressed by the tumor cells. We have generated a CAR specific for the splice variant extra domain A (EDA) of fibronectin, which is highly expressed in the tumor stroma of many types of tumors but not in healthy tissues. METHODS: EDA expression was explored in RNA-seq data from different human tumor types and by immunohistochemistry in paraffin-embedded tumor biopsies. Murine and human anti-EDA CAR-T cells were prepared using recombinant retro/lentiviruses, respectively. The functionality of EDA CAR-T cells was measured in vitro in response to antigen stimulation. The antitumor activity of EDA CAR-T cells was measured in vivo in C57BL/6 mice challenged with PM299L-EDA hepatocarcinoma cell line, in 129Sv mice-bearing F9 teratocarcinoma and in NSG mice injected with the human hepatocarcinoma cell line PLC. RESULTS: EDA CAR-T cells recognized and killed EDA-expressing tumor cell lines in vitro and rejected EDA-expressing tumors in immunocompetent mice. Notably, EDA CAR-T cells showed an antitumor effect in mice injected with EDA-negative tumor cells lines when the tumor stroma or the basement membrane of tumor endothelial cells express EDA. Thus, EDA CAR-T administration delayed tumor growth in immunocompetent 129Sv mice challenged with teratocarcinoma cell line F9. EDA CAR-T treatment exerted an antiangiogenic effect and significantly reduced gene signatures associated with epithelial-mesenchymal transition, collagen synthesis, extracellular matrix organization as well as IL-6-STAT5 and KRAS pathways. Importantly, the human version of EDA CAR, that includes the human 41BB and CD3ζ endodomains, exerted strong antitumor activity in NSG mice challenged with the human hepatocarcinoma cell line PLC, which expresses EDA in the tumor stroma and the endothelial vasculature. EDA CAR-T cells exhibited a tropism for EDA-expressing tumor tissue and no toxicity was observed in tumor bearing or in healthy mice. CONCLUSIONS: These results suggest that targeting the tumor-specific fibronectin splice variant EDA with CAR-T cells is feasible and offers a therapeutic option that is applicable to different types of cancer.

A Therapeutically Actionable Protumoral Axis of Cytokines Involving IL-8, TNFα, and IL-1ß.

Interleukin-8 (CXCL8) produced in the tumor microenvironment correlates with poor response to checkpoint inhibitors and is known to chemoattract and activate immunosuppressive myeloid leukocytes. In human cancer, IL8 mRNA levels correlate with IL1B and TNF transcripts. Both cytokines induced IL-8 functional expression from a broad variety of human cancer cell lines, primary colon carcinoma organoids, and fresh human tumor explants. Although IL8 is absent from the mouse genome, a similar murine axis in which TNFα and IL-1ß upregulate CXCL1 and CXCL2 in tumor cells was revealed. Furthermore, intratumoral injection of TNFα and IL-1ß induced IL-8 release from human malignant cells xenografted in immunodeficient mice. In all these cases, the clinically used TNFα blockers infliximab and etanercept or the IL-1ß inhibitor anakinra was able to interfere with this pathogenic cytokine loop. Finally, in paired plasma samples of patients with cancer undergoing TNFα blockade with infliximab in a clinical trial, reductions of circulating IL-8 were substantiated. SIGNIFICANCE: IL-8 attracts immunosuppressive protumor myeloid cells to the tumor microenvironment, and IL-8 levels correlate with poor response to checkpoint inhibitors. TNFα and IL-1ß are identified as major inducers of IL-8 expression on malignant cells across cancer types and models in a manner that is druggable with clinically available neutralizing agents. This article is highlighted in the In This Issue feature, p. 2007.

Neutrophil Extracellular Traps, Local IL-8 Expression, and Cytotoxic T-Lymphocyte Response in the Lungs of Patients With Fatal COVID-19.

BACKGROUND: Excessive inflammation is pathogenic in the pneumonitis associated with severe COVID-19. Neutrophils are among the most abundantly present leukocytes in the inflammatory infiltrates and may form neutrophil extracellular traps (NETs) under the local influence of cytokines. NETs constitute a defense mechanism against bacteria, but have also been shown to mediate tissue damage in a number of diseases. RESEARCH QUESTION: Could NETs and their tissue-damaging properties inherent to neutrophil-associated functions play a role in the respiratory failure seen in patients with severe COVID-19, and how does this relate to the SARS-CoV-2 viral loads, IL-8 (CXCL8) chemokine expression, and cytotoxic T-lymphocyte infiltrates? STUDY DESIGN AND METHODS: Sixteen lung biopsy samples obtained immediately after death were analyzed methodically as exploratory and validation cohorts. NETs were analyzed quantitatively by multiplexed immunofluorescence and were correlated with local levels of IL-8 messenger RNA (mRNA) and the density of CD8+ T-cell infiltration. SARS-CoV-2 presence in tissue was quantified by reverse-transcriptase polymerase chain reaction and immunohistochemistry analysis. RESULTS: NETs were found in the lung interstitium and surrounding the bronchiolar epithelium with interindividual and spatial heterogeneity. NET density did not correlate with SARS-CoV-2 tissue viral load. NETs were associated with local IL-8 mRNA levels. NETs were also detected in pulmonary thrombi and in only one of eight liver tissues. NET focal presence correlated negatively with CD8+ T-cell infiltration in the lungs. INTERPRETATION: Abundant neutrophils undergoing NETosis are found in the lungs of patients with fatal COVID-19, but no correlation was found with viral loads. The strong association between NETs and IL-8 points to this chemokine as a potentially causative factor. The function of cytotoxic T-lymphocytes in the immune responses against SARS-CoV-2 may be interfered with by the presence of NETs.

Exploiting 4-1BB immune checkpoint to enhance the efficacy of oncolytic virotherapy for diffuse intrinsic pontine gliomas.

Diffuse intrinsic pontine gliomas (DIPGs) are aggressive pediatric brain tumors, and patient survival has not changed despite many therapeutic efforts, emphasizing the urgent need for effective treatments. Here, we evaluated the anti-DIPG effect of the oncolytic adenovirus Delta-24-ACT, which was engineered to express the costimulatory ligand 4-1BBL to potentiate the antitumor immune response of the virus. Delta-24-ACT induced the expression of functional 4-1BBL on the membranes of infected DIPG cells, which enhanced the costimulation of CD8+ T lymphocytes. In vivo, Delta-24-ACT treatment of murine DIPG orthotopic tumors significantly improved the survival of treated mice, leading to long-term survivors that developed immunological memory against these tumors. In addition, Delta-24-ACT was safe and caused no local or systemic toxicity. Mechanistic studies showed that Delta-24-ACT modulated the tumor-immune content, not only increasing the number, but also improving the functionality of immune cells. All of these data highlight the safety and potential therapeutic benefit of Delta-24-ACT the treatment of patients with DIPG.