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.

Rs867228 in FPR1 accelerates the manifestation of luminal B breast cancer.

Formyl peptide receptor-1 (FPR1) is a pathogen recognition receptor involved in the detection of bacteria, in the control of inflammation, as well as in cancer immunosurveillance. A single nucleotide polymorphism in FPR1, rs867228, provokes a loss-of-function phenotype. In a bioinformatic study performed on The Cancer Genome Atlas (TCGA), we observed that homo-or heterozygosity for rs867228 in FPR1 (which affects approximately one-third of the population across continents) accelerates age at diagnosis of specific carcinomas including luminal B breast cancer by 4.9 years. To validate this finding, we genotyped 215 patients with metastatic luminal B mammary carcinomas from the SNPs To Risk of Metastasis (SToRM) cohort. The first diagnosis of luminal B breast cancer occurred at an age of 49.2 years for individuals bearing the dysfunctional TT or TG alleles (n = 73) and 55.5 years for patients the functional GG alleles (n = 141), meaning that rs867228 accelerated the age of diagnosis by 6.3 years (p=0.0077, Mann & Whitney). These results confirm our original observation in an independent validation cohort. We speculate that it may be useful to include the detection of rs867228 in breast cancer screening campaigns for selectively increasing the frequency and stringency of examinations starting at a relatively young age.

A loss-of-function polymorphism in ATG16L1 compromises therapeutic outcome in head and neck carcinoma patients.

The anticancer immune response is shaped by immunogenic cell stress and death pathways. Thus, cancer cells can release danger-associated molecular patterns that act on pattern recognition receptors expressed by dendritic cells and their precursors to elicit an antitumor immune response. Here, we investigated the impact of single nucleotide polymorphisms (SNPs) in genes affecting this cancer-immunity dialogue in the context of head and neck squamous cell carcinoma (HNSCC). We observed that homozygosity for a loss-of-function SNP (rs2241880, leading to the substitution of a threonine residue in position 300 by an alanine) affecting autophagy related 16 like 1 (ATG16L1) is coupled to poor progression-free survival in platinum-treated HNSCC patients. This result was obtained on a cohort of patients enrolled at the Gustave Roussy Cancer Campus and was validated on an independent cohort of The Cancer Genome Atlas (TCGA). Homozygosity in rs2241880 is well known to predispose to Crohn's disease, and epidemiological associations between Crohn's disease and HNSCC have been reported at the levels of cancer incidence and prognosis. We speculate that rs2241880 might be partially responsible for this association.

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.

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.

No impact of cancer and plague-relevant FPR1 polymorphisms on COVID-19.

Formyl peptide receptor 1 (FPR1) is a pattern-recognition receptor that detects bacterial as well as endogenous danger-associated molecular patterns to trigger innate immune responses by myeloid cells. A single nucleotide polymorphism, rs867228 (allelic frequency 19-20%), in the gene coding for FPR1 accelerates the manifestation of multiple carcinomas, likely due to reduced anticancer immunosurveillance secondary to a defect in antigen presentation by dendritic cells. Another polymorphism in FPR1, rs5030880 (allelic frequency 12-13%), has been involved in the resistance to plague, correlating with the fact that FPR1 is the receptor for Yersinia pestis. Driven by the reported preclinical effects of FPR1 on lung inflammation and fibrosis, we investigated whether rs867228 or rs5030880 would affect the severity of coronavirus disease-19 (COVID-19). Data obtained on patients from two different hospitals in Paris refute the hypothesis that rs867228 or rs5030880 would affect the severity of COVID-19.

Trial watch: TLR3 agonists in cancer therapy.

Toll-like receptor 3 (TLR3) is a pattern recognition receptor that senses exogenous (viral) as well as endogenous (mammalian) double-stranded RNA in endosomes. On activation, TLR3 initiates a signal transduction pathway that culminates with the secretion of pro-inflammatory cytokines including type I interferon (IFN). The latter is essential not only for innate immune responses to infection but also for the initiation of antigen-specific immunity against viruses and malignant cells. These aspects of TLR3 biology have supported the development of various agonists for use as stand-alone agents or combined with other therapeutic modalities in cancer patients. Here, we review recent preclinical and clinical advances in the development of TLR3 agonists for oncological disorders. Abbreviations: cDC, conventional dendritic cell; CMT, cytokine modulating treatment; CRC, colorectal carcinoma; CTL, cytotoxic T lymphocyte; DC, dendritic cell; dsRNA, double-stranded RNA; FLT3LG, fms-related receptor tyrosine kinase 3 ligand; HNSCC, head and neck squamous cell carcinoma; IFN, interferon; IL, interleukin; ISV, in situ vaccine; MUC1, mucin 1, cell surface associated; PD-1, programmed cell death 1; PD-L1, programmed death-ligand 1; polyA:U, polyadenylic:polyuridylic acid; polyI:C, polyriboinosinic:polyribocytidylic acid; TLR, Toll-like receptor.

Trial watch: STING agonists in cancer therapy.

Stimulator of interferon response cGAMP interactor 1 (STING1, best known as STING) is an endoplasmic reticulum-sessile protein that serves as a signaling hub, receiving input from several pattern recognition receptors, most of which sense ectopic DNA species in the cytosol. In particular, STING ensures the production of type I interferon (IFN) in response to invading DNA viruses, bacterial pathogens, as well as DNA leaking from mitochondria or the nucleus (e.g., in cells exposed to chemotherapy or radiotherapy). As a type I IFN is critical for the initiation of anticancer immune responses, the pharmaceutical industry has generated molecules that directly activate STING for use in oncological indications. Such STING agonists are being tested in clinical trials with the rationale of activating STING in tumor cells or tumor-infiltrating immune cells (including dendritic cells) to elicit immunostimulatory effects, alone or in combination with a range of established chemotherapeutic and immunotherapeutic regimens. In this Trial Watch, we discuss preclinical evidence and accumulating clinical experience shaping the design of Phase I and Phase II trials that evaluate the safety and preliminary efficacy of STING agonists in cancer patients.

Trial watch: IDO inhibitors in cancer therapy.

Indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the first, rate-limiting step of the so-called "kynurenine pathway", which converts the essential amino acid L-tryptophan (Trp) into the immunosuppressive metabolite L-kynurenine (Kyn). While expressed constitutively by some tissues, IDO1 can also be induced in specific subsets of antigen-presenting cells that ultimately favor the establishment of immune tolerance to tumor antigens. At least in part, the immunomodulatory functions of IDO1 can be explained by depletion of Trp and accumulation of Kyn and its derivatives. In animal tumor models, genetic or pharmacological IDO1 inhibition can cause the (re)activation of anticancer immune responses. Similarly, neoplasms expressing high levels of IDO1 may elude anticancer immunosurveillance. Therefore, IDO1 inhibitors represent promising therapeutic candidates for cancer therapy, and some of them have already entered clinical evaluation. Here, we summarize preclinical and clinical studies testing IDO1-targeting interventions for oncologic indications.

Trial Watch: experimental TLR7/TLR8 agonists for oncological indications.

Resiquimod (R848) and motolimod (VTX-2337) are second-generation experimental derivatives of imiquimod, an imidazoquinoline with immunostimulatory properties originally approved by the US Food and Drug Administration for the topical treatment of actinic keratosis and genital warts more than 20 years ago. Both resiquimod and motolimod operate as agonists of Toll-like receptor 7 (TLR7) and/or TLR8, in thus far delivering adjuvant-like signals to antigen-presenting cells (APCs). In line with such an activity, these compounds are currently investigated as immunostimulatory agents for the treatment of various malignancies, especially in combination with peptide-based, dendritic cell-based, cancer cell lysate-based, or DNA-based vaccines. Here, we summarize preclinical and clinical evidence recently collected to support the development of resiquimod and motolimod and other TLR7/TLR8 agonists as anticancer agents.

Chemical activation of SAT1 corrects diet-induced metabolic syndrome.

The pharmacological targeting of polyamine metabolism is currently under the spotlight for its potential in the prevention and treatment of several age-associated disorders. Here, we report the finding that triethylenetetramine dihydrochloride (TETA), a copper-chelator agent that can be safely administered to patients for the long-term treatment of Wilson disease, exerts therapeutic benefits in animals challenged with hypercaloric dietary regimens. TETA reduced obesity induced by high-fat diet, excessive sucrose intake, or leptin deficiency, as it reduced glucose intolerance and hepatosteatosis, but induced autophagy. Mechanistically, these effects did not involve the depletion of copper from plasma or internal organs. Rather, the TETA effects relied on the activation of an energy-consuming polyamine catabolism, secondary to the stabilization of spermidine/spermine N1-acetyltransferase-1 (SAT1) by TETA, resulting in enhanced enzymatic activity of SAT. All the positive effects of TETA on high-fat diet-induced metabolic syndrome were lost in SAT1-deficient mice. Altogether, these results suggest novel health-promoting effects of TETA that might be taken advantage of for the prevention or treatment of obesity.

A synergistic triad of chemotherapy, immune checkpoint inhibitors, and caloric restriction mimetics eradicates tumors in mice.

We have recently shown that chemotherapy with immunogenic cell death (ICD)-inducing agents can be advantageously combined with fasting regimens or caloric restriction mimetics (CRMs) to achieve superior tumor growth control via a T cell-dependent mechanism. Here, we show that the blockade of the CD11b-dependent extravasation of myeloid cells blocks such a combination effect as well. Based on the characterization of the myeloid and lymphoid immune infiltrates, including the expression pattern of immune checkpoint proteins (and noting a chemotherapy-induced overexpression of programmed death-ligand 1, PD-L1, on both cancer cells and leukocytes, as well as a reduced frequency of exhausted CD8+ T cells positive for programmed cell death 1 protein, PD-1), we then evaluated the possibility to combine ICD inducers, CRMs and targeting of the PD-1/PD-L1 interaction. While fasting or CRMs failed to improve tumor growth control by PD-1 blockade, ICD inducers alone achieved a partial sensitization to treatment with a PD-1-specific antibody. However, definitive cure of most of the tumor-bearing mice was only achieved by a tritherapy combining (i) ICD inducers exemplified by mitoxantrone and oxaliplatin, (ii) CRMs exemplified by hydroxycitrate and spermidine and substitutable for by fasting, and (iii) immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 interaction. Altogether, these results point to the possibility of synergistic interactions among distinct classes of anticancer agents.

Crizotinib-induced immunogenic cell death in non-small cell lung cancer.

Immunogenic cell death (ICD) converts dying cancer cells into a therapeutic vaccine and stimulates antitumor immune responses. Here we unravel the results of an unbiased screen identifying high-dose (10 µM) crizotinib as an ICD-inducing tyrosine kinase inhibitor that has exceptional antineoplastic activity when combined with non-ICD inducing chemotherapeutics like cisplatin. The combination of cisplatin and high-dose crizotinib induces ICD in non-small cell lung carcinoma (NSCLC) cells and effectively controls the growth of distinct (transplantable, carcinogen- or oncogene induced) orthotopic NSCLC models. These anticancer effects are linked to increased T lymphocyte infiltration and are abolished by T cell depletion or interferon-γ neutralization. Crizotinib plus cisplatin leads to an increase in the expression of PD-1 and PD-L1 in tumors, coupled to a strong sensitization of NSCLC to immunotherapy with PD-1 antibodies. Hence, a sequential combination treatment consisting in conventional chemotherapy together with crizotinib, followed by immune checkpoint blockade may be active against NSCLC.

Author Correction: Crizotinib-induced immunogenic cell death in non-small cell lung cancer.

The original version of this Article contained an error in the spelling of the author Lin Xia, which was incorrectly given as Xia Lin. This has now been corrected in both the PDF and HTML versions of the Article.

Contribution of annexin A1 to anticancer immunosurveillance.

Mouse cancers lacking the expression of annexin A1 (ANXA1) fail to respond to immunogenic chemotherapies. This has been initially explained by the requirement of extracellular ANXA1 (which is released from dying cancer cells) to engage formyl peptide receptor-1 (FPR1) on dendritic cells (DC) for the establishment of corpse/DC synapses. Here, we show that ANXA1-deficent cancer cells exhibit a defect in the exposure of calreticulin (CALR), which is an important "eat-me" signal, facilitating the phagocytic uptake of dead-cell antigens by DC. Of note, intratumoral injection of recombinant CALR protein was able to restore the therapeutic response of ANXA1-deficient cancers to anthracycline-based chemotherapy. Carcinomas developing in patients tend to downregulate ANXA1 expression as compared to their normal tissues of origin. ANXA1-low breast, colorectal, lung and kidney cancers are scarcely infiltrated by DC and cytotoxic T lymphocytes, supporting the idea that ANXA1 deficiency facilitates immune escape. We propose that such ANXA1-low cancers might be particularly suitable to local immunotherapy with CALR protein.

Systemic autophagy in the therapeutic response to anthracycline-based chemotherapy.

The success of chemotherapy largely depends on the anticancer immune response triggered by tumor cells that succumb to immunogenic cell death (ICD). One of the hallmarks of ICD is premortem autophagy that facilitates the release of adenosine triphosphate from dying cancer cells and acts as a chemoattractant for dendritic cell precursors. Here, we show that the immune response induced by inoculation of cancer cells undergoing ICD in response to the anthracycline mitoxantrone (MTX) can be improved by a short-term fasting regimen (48 hours of starvation) and that this effect is reversed by systemic administration of the autophagy inhibitor dimethyl α-ketoglutarate. Tumor growth reduction by MTX treatment is known to depend on autophagy induction in cancer cells as well as on an intact immune system. We compared the antitumor effects of MTX on autophagy-competent cancers implanted in wild type (WT) or partially autophagy-deficient (Becn1± or Atg4b-/-) mice. While there was no difference in the tumor growth reducing effects of MTX on tumors evolving in WT, Becn1+/- and Atg4b-/- mice, we observed an increase in the toxicity of MTX on Atg4b-/- mice. These results suggest that autophagy in cancer cells (but less so in host cells) is rate-limiting for therapeutically relevant anticancer immune responses, yet has a major role in blunting the life-threatening toxicity of chemotherapy.

TumGrowth: An open-access web tool for the statistical analysis of tumor growth curves.

The analysis of tumor growth curves is standard practice in experimental oncology including tumor immunology. In experimental oncology, cancer cells are inoculated into rodents (mostly mice) and their growth is monitored by measuring tumor diameter, surface or volume over time as a function of distinct treatments. Then, different groups of tumors/treatments are compared among each other for their evolution and possible responses to treatment. The R package TumGrowth has been created as a software tool allowing to carry out a series of statistical comparisons across or between groups of tumor growth curves obtained in a standard laboratory, for experimenters with limited knowledge in statistics. TumGrowth is freely available online at https://kroemerlab.shinyapps.io/TumGrowth/ and can be downloaded into any computer. It offers an exhaustive panoply of tools to visualize and analyze complex data sets including longitudinal, cross-sectional and time-to-endpoint measurements.