Tutorial: design, production and testing of oncolytic viruses for cancer immunotherapy.

Oncolytic viruses (OVs) represent a novel class of cancer immunotherapy agents that preferentially infect and kill cancer cells and promote protective antitumor immunity. Furthermore, OVs can be used in combination with established or upcoming immunotherapeutic agents, especially immune checkpoint inhibitors, to efficiently target a wide range of malignancies. The development of OV-based therapy involves three major steps before clinical evaluation: design, production and preclinical testing. OVs can be designed as natural or engineered strains and subsequently selected for their ability to kill a broad spectrum of cancer cells rather than normal, healthy cells. OV selection is further influenced by multiple factors, such as the availability of a specific viral platform, cancer cell permissivity, the need for genetic engineering to render the virus non-pathogenic and/or more effective and logistical considerations around the use of OVs within the laboratory or clinical setting. Selected OVs are then produced and tested for their anticancer potential by using syngeneic, xenograft or humanized preclinical models wherein immunocompromised and immunocompetent setups are used to elucidate their direct oncolytic ability as well as indirect immunotherapeutic potential in vivo. Finally, OVs demonstrating the desired anticancer potential progress toward translation in patients with cancer. This tutorial provides guidelines for the design, production and preclinical testing of OVs, emphasizing considerations specific to OV technology that determine their clinical utility as cancer immunotherapy agents.

Immunogenic oncolysis by tigilanol tiglate.

Tigilanol tiglate is an oncolytic small molecule that is undergoing clinical trials. A recent study revealed the capacity of this pyroptosis inducer to elicit hallmarks of immunogenic cell death. In addition, intratumoral injection of tigilanol tiglate can sensitize subcutaneous cancers to subsequent immune checkpoint inhibitors targeting CTLA-4 alone or in combination with PD-1.

Single-cell analysis of T lymphocytes infiltrating colorectal carcinoma: the dilemma of specificity.

Advances in single-cell RNA and T cell receptor (TCR) sequencing allow to study the specificity and functionality of tumor-infiltrating T lymphocytes. A recent study unravels fundamental differences between microsatellite-instable (MSI) colorectal cancers, in which T cells tend to be tumor-specific, and microsatellite-stable (MSS) cancers, in which T cells exhibit bystander features.

Anticancer effects of ikarugamycin and astemizole identified in a screen for stimulators of cellular immune responses.

BACKGROUND: Most immunotherapies approved for clinical use rely on the use of recombinant proteins and cell-based approaches, rendering their manufacturing expensive and logistics onerous. The identification of novel small molecule immunotherapeutic agents might overcome such limitations. METHOD: For immunopharmacological screening campaigns, we built an artificial miniature immune system in which dendritic cells (DCs) derived from immature precursors present MHC (major histocompatibility complex) class I-restricted antigen to a T-cell hybridoma that then secretes interleukin-2 (IL-2). RESULTS: The screening of three drug libraries relevant to known signaling pathways, FDA (Food and Drug Administration)-approved drugs and neuroendocrine factors yielded two major hits, astemizole and ikarugamycin. Mechanistically, ikarugamycin turned out to act on DCs to inhibit hexokinase 2, hence stimulating their antigen presenting potential. In contrast, astemizole acts as a histamine H1 receptor (H1R1) antagonist to activate T cells in a non-specific, DC-independent fashion. Astemizole induced the production of IL-2 and interferon-γ (IFN-γ) by CD4+ and CD8+ T cells both in vitro and in vivo. Both ikarugamycin and astemizole improved the anticancer activity of the immunogenic chemotherapeutic agent oxaliplatin in a T cell-dependent fashion. Of note, astemizole enhanced the CD8+/Foxp3+ ratio in the tumor immune infiltrate as well as IFN-γ production by local CD8+ T lymphocytes. In patients with cancer, high H1R1 expression correlated with low infiltration by TH1 cells, as well as with signs of T-cell exhaustion. The combination of astemizole and oxaliplatin was able to cure the majority of mice bearing orthotopic non-small cell lung cancers (NSCLC), then inducing a state of protective long-term immune memory. The NSCLC-eradicating effect of astemizole plus oxaliplatin was lost on depletion of either CD4+ or CD8+ T cells, as well as on neutralization of IFN-γ. CONCLUSIONS: These findings underscore the potential utility of this screening system for the identification of immunostimulatory drugs with anticancer effects.

Formyl peptide receptor-1 (FPR1) represses intestinal oncogenesis.

Formyl peptide receptor-1 (FPR1) is a pattern recognition receptor that is mostly expressed by myeloid cells. In patients with colorectal cancer (CRC), a loss-of-function polymorphism (rs867228) in the gene coding for FPR1 has been associated with reduced responses to chemotherapy or chemoradiotherapy. Moreover, rs867228 is associated with accelerated esophageal and colorectal carcinogenesis. Here, we show that dendritic cells from Fpr1-/- mice exhibit reduced migration in response to chemotherapy-treated CRC cells. Moreover, Fpr1-/- mice are particularly susceptible to chronic ulcerative colitis and colorectal oncogenesis induced by the mutagen azoxymethane followed by oral dextran sodium sulfate, a detergent that induces colitis. These experiments were performed after initial co-housing of Fpr1-/- mice and wild-type controls, precluding major Fpr1-driven differences in the microbiota. Pharmacological inhibition of Fpr1 by cyclosporin H also tended to increase intestinal oncogenesis in mice bearing the ApcMin mutation, and this effect was reversed by the anti-inflammatory drug sulindac. We conclude that defective FPR1 signaling favors intestinal tumorigenesis through the modulation of the innate inflammatory/immune response.

Systematic Investigation of the Diagnostic and Prognostic Impact of LINC01087 in Human Cancers.

(1) Background: Long non-coding RNAs may constitute epigenetic biomarkers for the diagnosis, prognosis, and therapeutic response of a variety of tumors. In this context, we aimed at assessing the diagnostic and prognostic value of the recently described long intergenic non-coding RNA 01087 (LINC01087) in human cancers. (2) Methods: We studied the expression of LINC01087 across 30 oncological indications by interrogating public resources. Data extracted from the TCGA and GTEx databases were exploited to plot receiver operating characteristic curves (ROC) and determine the diagnostic performance of LINC01087. Survival data from TCGA and KM-Plotter directories allowed us to graph Kaplan-Meier curves and evaluate the prognostic value of LINC01087. To investigate the function of LINC01087, gene ontology (GO) annotation and Kyoto Encyclopedia of Gene and Genomes (KEGG) enrichment analyses were performed. Furthermore, interactions between LINC01087 and both miRNA and mRNA were studied by means of bioinformatics tools. (3) Results: LINC01087 was significantly deregulated in 7 out of 30 cancers, showing a predominant upregulation. Notably, it was overexpressed in breast (BC), esophageal (ESCA), and ovarian (OV) cancers, as well as lung squamous cell carcinoma (LUSC), stomach adenocarcinoma (STAD), and uterine carcinosarcoma (UCS). By contrast, LINC01087 displayed downregulation in testicular germ cell tumors (TGCT). ROC curve analyses identified LINC01087 as a potential diagnostic indicator in BC, ESCA, OV, STAD, and TGCT. Moreover, high and low expression of LINC01087 predicted a favorable prognosis in BC and papillary cell carcinoma, respectively. In silico analyses indicated that deregulation of LINC01087 in cancer was associated with a modulation of genes related to ion channel, transporter, and peptide receptor activity. (4) Conclusions: the quantification of an altered abundance of LINC01087 in tissue specimens might be clinically useful for the diagnosis and prognosis of some hormone-related tumors, including BC, OV, and TGCT, as well as other cancer types such as ESCA and STAD. Moreover, our study revealed the potential of LINC01087 (and perhaps other lncRNAs) to regulate neuroactive molecules in cancer.

Improved Swiss-rolling method for histological analyses of colon tissue.

Since the introduction of the Swiss-rolling technique by Reilly and Kirsner in 1965, various methodological approaches have been developed for histological analyses of intestinal tissues. Here, we describe an improved protocol for the processing of freshly harvested murine colons that can be extended to other intestinal tissues. With simple tools, this technique allows to tightly wrap the organ throughout the whole length and to keep it in place before fixation, avoiding excessive stiffness of the tissue. Unlike the original method which relies on frozen samples, processing of the biological samples right after resection preserves epitopes integrity for subsequent immunohistochemical analyses. Ultimately, this method provides a reproducible workflow to capture the entire colon length in a unique histological section in order to assess several features such as intestinal inflammation and tumorigenesis. • Easily include freshly isolated tissues • Shorten preparation time using a few affordable tools • Prevent unrolling and preserve tissue integrity.

Circular RNAs as Potential Biomarkers in Breast Cancer.

Due to the high heterogeneity and initially asymptomatic nature of breast cancer (BC), the management of this disease depends on imaging together with immunohistochemical and molecular evaluations. These tests allow early detection of BC and patient stratification as they guide clinicians in prognostication and treatment decision-making. Circular RNAs (circRNAs) represent a class of newly identified long non-coding RNAs. These molecules have been described as key regulators of breast carcinogenesis and progression. Moreover, circRNAs play a role in drug resistance and are associated with clinicopathological features in BC. Accumulating evidence reveals a clinical interest in deregulated circRNAs as diagnostic, prognostic and predictive biomarkers. Furthermore, due to their covalently closed structure, circRNAs are highly stable and easily detectable in body fluids, making them ideal candidates for use as non-invasive biomarkers. Herein, we provide an overview of the biogenesis and pleiotropic functions of circRNAs, and report on their clinical relevance in BC.

Trial watch: intratumoral immunotherapy.

While chemotherapy and radiotherapy remain the first-line approaches for the management of most unresectable tumors, immunotherapy has emerged in the past two decades as a game-changing treatment, notably with the clinical success of immune checkpoint inhibitors. Immunotherapies aim at (re)activating anticancer immune responses which occur in two main steps: (1) the activation and expansion of tumor-specific T cells following cross-presentation of tumor antigens by specialized myeloid cells (priming phase); and (2) the immunological clearance of malignant cells by these antitumor T lymphocytes (effector phase). Therapeutic vaccines, adjuvants, monoclonal antibodies, cytokines, immunogenic cell death-inducing agents including oncolytic viruses, anthracycline-based chemotherapy and radiotherapy, as well as adoptive cell transfer, all act at different levels of this cascade to (re)instate cancer immunosurveillance. Intratumoral delivery of these immunotherapeutics is being tested in clinical trials to promote superior antitumor immune activity in the context of limited systemic toxicity.

Autoimmunity affecting the biliary tract fuels the immunosurveillance of cholangiocarcinoma.

Cholangiocarcinoma (CCA) results from the malignant transformation of cholangiocytes. Primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) are chronic diseases in which cholangiocytes are primarily damaged. Although PSC is an inflammatory condition predisposing to CCA, CCA is almost never found in the autoimmune context of PBC. Here, we hypothesized that PBC might favor CCA immunosurveillance. In preclinical murine models of cholangitis challenged with syngeneic CCA, PBC (but not PSC) reduced the frequency of CCA development and delayed tumor growth kinetics. This PBC-related effect appeared specific to CCA as it was not observed against other cancers, including hepatocellular carcinoma. The protective effect of PBC was relying on type 1 and type 2 T cell responses and, to a lesser extent, on B cells. Single-cell TCR/RNA sequencing revealed the existence of TCR clonotypes shared between the liver and CCA tumor of a PBC host. Altogether, these results evidence a mechanistic overlapping between autoimmunity and cancer immunosurveillance in the biliary tract.

Beneficial autoimmunity links primary biliary cholangitis to the avoidance of cholangiocarcinoma.

It has been an open conundrum why primary sclerosing cholangitis (PSC) is a major risk factor for developing cholangiocarcinoma (CAA), while primary biliary cholangitis (PBC) is not. In mouse models of PSC and PBC, it turned out that the latter condition, an autoimmune disease affecting the bile ducts, reduces transgene-induced cholangiocarcinogenesis, as well as the progression of subcutaneously implanted CCA. This CCA-delaying effect is lost upon depletion of T lymphocytes and involves tumor infiltration by T cell clonotypes that are also found in PBC lesions. Hence, organ-specific autoimmunity may improve immunosurveillance.

Metabolic Reprogramming by Reduced Calorie Intake or Pharmacological Caloric Restriction Mimetics for Improved Cancer Immunotherapy.

Caloric restriction and fasting have been known for a long time for their health- and life-span promoting effects, with coherent observations in multiple model organisms as well as epidemiological and clinical studies. This holds particularly true for cancer. The health-promoting effects of caloric restriction and fasting are mediated at least partly through their cellular effects-chiefly autophagy induction-rather than reduced calorie intake per se. Interestingly, caloric restriction has a differential impact on cancer and healthy cells, due to the atypical metabolic profile of malignant tumors. Caloric restriction mimetics are non-toxic compounds able to mimic the biochemical and physiological effects of caloric restriction including autophagy induction. Caloric restriction and its mimetics induce autophagy to improve the efficacy of some cancer treatments that induce immunogenic cell death (ICD), a type of cellular demise that eventually elicits adaptive antitumor immunity. Caloric restriction and its mimetics also enhance the therapeutic efficacy of chemo-immunotherapies combining ICD-inducing agents with immune checkpoint inhibitors targeting PD-1. Collectively, preclinical data encourage the application of caloric restriction and its mimetics as an adjuvant to immunotherapies. This recommendation is subject to confirmation in additional experimental settings and in clinical trials. In this work, we review the preclinical and clinical evidence in favor of such therapeutic interventions before listing ongoing clinical trials that will shed some light on this subject.

A TLR3 Ligand Reestablishes Chemotherapeutic Responses in the Context of FPR1 Deficiency.

For anthracycline-based chemotherapy to be immunogenic, dying cancer cells must release annexin A1 (ANXA1) that subsequently interacts with the pattern recognition receptor, formyl peptide receptor 1 (FPR1), on the surface of dendritic cells (DC). Approximately 30% of individuals bear loss-of-function alleles of FPR1, calling for strategies to ameliorate their anticancer immune response. Here, we show that immunotherapy with a ligand of Toll-like receptor-3, polyinosinic:polycytidylic acid (pIC), restores the deficient response to chemotherapy of tumors lacking ANXA1 developing in immunocompetent mice or those of normal cancers growing in FPR1-deficient mice. This effect was accompanied by improved DC- and T-lymphocyte-mediated anticancer immunity. Of note, carcinogen-induced breast cancers precociously developed in FPR1-deficient mice as compared with wild-type controls. A similar tendency for earlier cancer development was found in patients carrying the loss-of-function allele of FPR1. These findings have potential implications for the clinical management of FPR1-deficient patients. SIGNIFICANCE: The loss-of-function variant rs867228 in FPR1, harbored by approximately 30% of the world population, is associated with the precocious manifestation of breast, colorectal, esophageal, and head and neck carcinomas. pIC restores deficient chemotherapeutic responses in mice lacking Fpr1, suggesting a personalized strategy for compensating for the FPR1 defect.This article is highlighted in the In This Issue feature, p. 211.

Autophagy-mediated metabolic effects of aspirin.

Salicylate, the active derivative of aspirin (acetylsalicylate), recapitulates the mode of action of caloric restriction inasmuch as it stimulates autophagy through the inhibition of the acetyltransferase activity of EP300. Here, we directly compared the metabolic effects of aspirin medication with those elicited by 48 h fasting in mice, revealing convergent alterations in the plasma and the heart metabolome. Aspirin caused a transient reduction of general protein acetylation in blood leukocytes, accompanied by the induction of autophagy. However, these effects on global protein acetylation could not be attributed to the mere inhibition of EP300, as determined by epistatic experiments and exploration of the acetyl-proteome from salicylate-treated EP300-deficient cells. Aspirin reduced high-fat diet-induced obesity, diabetes, and hepatosteatosis. These aspirin effects were observed in autophagy-competent mice but not in two different models of genetic (Atg4b-/- or Bcln1+/-) autophagy-deficiency. Aspirin also improved tumor control by immunogenic chemotherapeutics, and this effect was lost in T cell-deficient mice, as well as upon knockdown of an essential autophagy gene (Atg5) in cancer cells. Hence, the health-improving effects of aspirin depend on autophagy.

Dynamical Boolean Modeling of Immunogenic Cell Death.

As opposed to the standard tolerogenic apoptosis, immunogenic cell death (ICD) constitutes a type of cellular demise that elicits an adaptive immune response. ICD has been characterized in malignant cells following cytotoxic interventions, such as chemotherapy or radiotherapy. Briefly, ICD of cancer cells releases some stress/danger signals that attract and activate dendritic cells (DCs). The latter can then engulf and cross-present tumor antigens to T lymphocytes, thus priming a cancer-specific immunity. This series of reactions works as a positive feedback loop where the antitumor immunity further improves the therapeutic efficacy by targeting cancer cells spared by the cytotoxic agent. However, not all chemotherapeutic drugs currently approved for cancer treatment are able to stimulate bona fide ICD: some commonly used agents, such as cisplatin or 5-fluorouracil, are unable to activate all features of ICD. Therefore, a better characterization of the process could help identify some gene or protein candidates to target pharmacologically and suggest combinations of drugs that would favor/increase antitumor immune response. To this end, we have built a mathematical model of the major cell types that intervene in ICD, namely cancer cells, DCs, CD8+ and CD4+ T cells. Our model not only integrates intracellular mechanisms within each individual cell entity, but also incorporates intercellular communications between them. The resulting cell population model recapitulates key features of the dynamics of ICD after an initial treatment, in particular the time-dependent size of the different cell types. The model is based on a discrete Boolean formalism and is simulated by means of a software tool, UPMaBoSS, which performs stochastic simulations with continuous time, considering the dynamics of the system at the cell population level with appropriate timing of events, and accounting for death and division of each cell type. With this model, the time scales of some of the processes involved in ICD, which are challenging to measure experimentally, have been predicted. In addition, our model analysis led to the identification of actionable targets for boosting ICD-induced antitumor response. All computational analyses and results are compiled in interactive notebooks which cover the presentation of the network structure, model simulations, and parameter sensitivity analyses.

Cytokines in oncolytic virotherapy.

Tumors represent a hostile environment for the effector cells of cancer immunosurveillance. Immunosuppressive receptors and soluble or membrane-bound ligands are abundantly exposed and released by malignant entities and their stromal accomplices. As a consequence, executioners of antitumor immunity inefficiently navigate across cancer tissues and fail to eliminate malignant targets. By inducing immunogenic cancer cell death, oncolytic viruses profoundly reshape the tumor microenvironment. They trigger the local spread of danger signals and tumor-associated (as well as viral) antigens, thus attracting antigen-presenting cells, promoting the activation and expansion of lymphocytic populations, facilitating their infiltration in the tumor bed, and reinvigorating cytotoxic immune activity. The present review recapitulates key chemokines, growth factors and other cytokines that orchestrate this ballet of antitumoral leukocytes upon oncolytic virotherapy.

The abundance of the long intergenic non-coding RNA 01087 differentiates between luminal and triple-negative breast cancers and predicts patient outcome.

The molecular complexity of human breast cancer (BC) renders the clinical management of the disease challenging. Long non-coding RNAs (lncRNAs) are promising biomarkers for BC patient stratification, early detection, and disease monitoring. Here, we identified the involvement of the long intergenic non-coding RNA 01087 (LINC01087) in breast oncogenesis. LINC01087 appeared significantly downregulated in triple-negative BCs (TNBCs) and upregulated in the luminal BC subtypes in comparison to mammary samples from cancer-free women and matched normal cancer pairs. Interestingly, deregulation of LINC01087 allowed to accurately distinguish between luminal and TNBC specimens, independently of the clinicopathological parameters, and of the histological and TP53 or BRCA1/2 mutational status. Moreover, increased expression of LINC01087 predicted a better prognosis in luminal BCs, while TNBC tumors that harbored lower levels of LINC01087 were associated with reduced relapse-free survival. Furthermore, bioinformatics analyses were performed on TNBC and luminal BC samples and suggested that the putative tumor suppressor activity of LINC01087 may rely on interferences with pathways involved in cell survival, proliferation, adhesion, invasion, inflammation and drug sensitivity. Altogether, these data suggest that the assessment of LINC01087 deregulation could represent a novel, specific and promising biomarker not only for the diagnosis and prognosis of luminal BC subtypes and TNBCs, but also as a predictive biomarker of pharmacological interventions.

Publisher Correction: Immunoprophylactic and immunotherapeutic control of hormone receptor-positive breast cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Repurposing CD8+ T cell immunity against SARS-CoV-2 for cancer immunotherapy: a positive aspect of the COVID-19 pandemic?

The COVID-19 pandemic has afflicted most countries on the planet. As a result, immunity against SARS-CoV-2, induced via natural infections or imminent vaccinations, is expected to develop in a large fraction of the global population. Here, we propose to exploit SARS-CoV-2-specific CD8+ T cells for cancer immunotherapy strategies.