Macrophages Impair TLR9 Agonist Antitumor Activity through Interacting with the Anti-PD-1 Antibody Fc Domain.

BACKGROUND: A combination of TLR9 agonists and an anti-PD-1 antibody has been reported to be effective in immunocompetent mice but the role of innate immunity has not yet been completely elucidated. Therefore, we investigated the contribution of the innate immune system to this combinatorial immunotherapeutic regimens using an immunodeficient mouse model in which the effector functions of innate immunity can clearly emerge without any interference from T lymphocytes. METHODS: Athymic mice xenografted with IGROV-1 human ovarian cells, reported to be sensitive to TLR9 agonist therapy, were treated with cytosine-guanine (CpG)-oligodeoxynucleotides (ODNs), an anti-PD-1 antibody or their combination. RESULTS: We found that PD-1 blockade dampened CpG-ODN antitumor activity. In vitro studies indicated that the interaction between the anti-PD-1 antibody fragment crystallizable (Fc) domain and macrophage Fc receptors caused these immune cells to acquire an immunoregulatory phenotype, contributing to a decrease in the efficacy of CpG-ODNs. Accordingly, in vivo macrophage depletion abrogated the detrimental effect exerted by the anti-PD-1 antibody. CONCLUSION: Our data suggest that if TLR signaling is active in macrophages, coadministration of an anti-PD-1 antibody can reprogram these immune cells towards a polarization state able to negatively affect the immune response and eventually promote tumor growth.

Aerosol 1,25-dihydroxyvitamin D3 supplementation: A strategy to boost anti-tumor innate immune activity.

BACKGROUND: 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] plays a role in calcium homeostasis but can also exert immunomodulatory effects. In lungs, characterized by a particular immunosuppressive environment primarily due to the presence of alveolar macrophages (AM), 1,25(OH)2D3 has been shown to favor the immune response against pathogens. Here, we explored the ability of aerosolized 1,25(OH)2D3 to locally promote an anti-tumor phenotype in alveolar macrophages (AM) in the treatment of lung metastases. METHODS: Cytotoxicity assay has been used to assess the capability of AM, in vitro treated of not with 1,25(OH)2D3, to stimulate NK cells. Sulforhodamine B (SRB) assay has been used to assess the effect of 1,25(OH)2D3 on MC-38 and B16 tumor cells in vitro growth. 1,25(OH)2D3 was aerosolized in immunocompetent mouse models to evaluate the effect of local administration of 1,25(OH)2D3 on in vivo growth of MC-38 and B16 tumor cells within lungs and on infiltrating immune cells. RESULTS: In vitro incubation of naïve AM with 1,25(OH)2D3 improved their ability to stimulate NK cell cytotoxicity. In vivo aerosolized 1,25(OH)2D3 significantly reduced the metastatic growth of MC-38 colon carcinoma, a tumor histotype that frequently metastasizes to lung in human. Immune infiltrate obtained from digested lungs of 1,25(OH)2D3-treated mice bearing MC-38 metastases revealed an increased expression of MHCII and CD80 on AM and an up-modulation of CD69 expression on effector cells that paralleled a strong increased ability of these cells to kill MC-38 tumor in vitro. CONCLUSIONS: Together, these data show that aerosol delivery can represent a feasible and novel approach to supplement 1,25(OH)2D3 directly to the lungs promoting the activation of local immunity against cancer.

Gut Microbiota Condition the Therapeutic Efficacy of Trastuzumab in HER2-Positive Breast Cancer.

Emerging evidence indicates that gut microbiota affect the response to anticancer therapies by modulating the host immune system. In this study, we investigated the impact of gut microbiota on immune-mediated trastuzumab antitumor efficacy in preclinical models of HER2-positive breast cancer and in 24 patients with primary HER2-positive breast cancer undergoing trastuzumab-containing neoadjuvant treatment. In mice, the antitumor activity of trastuzumab was impaired by antibiotic administration or fecal microbiota transplantation from antibiotic-treated donors. Modulation of the intestinal microbiota was reflected in tumors by impaired recruitment of CD4+ T cells and granzyme B-positive cells after trastuzumab treatment. Antibiotics caused reductions in dendritic cell (DC) activation and the release of IL12p70 upon trastuzumab treatment, a mechanism that was necessary for trastuzumab effectiveness in our model. In patients, lower α-diversity and lower abundance of Lachnospiraceae, Turicibacteraceae, Bifidobacteriaceae, and Prevotellaceae characterized nonresponsive patients (NR) compared with those who achieved pathologic complete response (R), similar to antibiotic-treated mice. The transfer of fecal microbiota from R and NR into mice bearing HER2-positive breast cancer recapitulated the response to trastuzumab observed in patients. Fecal microbiota ß-diversity segregated patients according to response and positively correlated with immune signature related to interferon (IFN) and NO2-IL12 as well as activated CD4+ T cells and activated DCs in tumors. Overall, our data reveal the direct involvement of the gut microbiota in trastuzumab efficacy, suggesting that manipulation of the gut microbiota is an optimal future strategy to achieve a therapeutic effect or to exploit its potential as a biomarker for treatment response. SIGNIFICANCE: Evidence of gut microbiota involvement in trastuzumab efficacy represents the foundation for new therapeutic strategies aimed at manipulating commensal bacteria to improve response in trastuzumab-resistant patients.See related commentary by Sharma, p. 1937 GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/2195/F1.large.jpg.

Mechanisms of hyperprogressive disease after immune checkpoint inhibitor therapy: what we (don't) know.

Immune checkpoint inhibitors (ICIs) have made a breakthrough in the treatment of different types of tumors, leading to improvement in survival, even in patients with advanced cancers. Despite the good clinical results, a certain percentage of patients do not respond to this kind of immunotherapy. In addition, in a fraction of nonresponder patients, which can vary from 4 to 29% according to different studies, a paradoxical boost in tumor growth after ICI administration was observed: a completely unpredictable novel pattern of cancer progression defined as hyperprogressive disease. Since this clinical phenomenon has only been recently described, a universally accepted clinical definition is lacking, and major efforts have been made to uncover the biological bases underlying hyperprogressive disease. The lines of research pursued so far have focused their attention on the study of the immune tumor microenvironment or on the analysis of intrinsic genomic characteristics of cancer cells producing data that allowed us to formulate several hypotheses to explain this detrimental effect related to ICI therapy. The aim of this review is to summarize the most important works that, to date, provide important insights that are useful in understanding the mechanistic causes of hyperprogressive disease.

Combined targeting of EGFR and HER2 against prostate cancer stem cells.

Progression of prostate cancer has been associated with EGFR and HER2 activation and to tumor-initiating cells contribution toward chemotherapy resistance. We investigated the efficacy of a dual intervention against EGFR and HER2 to deplete the tumor-initiating cells, optimize the chemotherapy management and prevent the progression of castration-resistant prostate cancer (CRPC) cells. Using DU145, PC3, and 22Rv1 CRPC cell lines, biochemical analysis revealed activation of EGFR, HER2, MAPK, and STAT3 in DU145 and 22Rv1, and AKT and SRC in DU145 and PC-3. pSTAT3 nuclear staining was observed in DU145 xenografts and in 12 out of 14 CRPC specimens. The in vivo dual targeting of ErbB receptors with Cetuximab and Trastuzumab combined with chemotherapy caused an effective antitumor response in DU145 xenografted mice displaying STAT3 activation; conversely PC-3 bearing mice experienced tumor relapse. The potentiating of in vivo cytotoxic effect in DU145 model was accompanied by a significant decrease of prostatosphere-forming capacity assessed in vitro on residual tumor cells. Additionally, combined treatment in vitro with Cetuximab, Trastuzumab and chemotherapy negatively affected DU145 and 22Rv1 sphere formation, suggesting the critical function of ErbB receptors for tumor-initiating cells proliferation; no effect on PC-3 clonogenic potential was observed, indicating that other receptors than EGFR and HER2 may sustain PC3 tumor-initiating cells. These findings provided the preclinical evidence that the dual inhibition of EGFR and HER2 by targeting tumor-initiating cells may improve the efficacy of the current chemotherapy regimen, bringing benefits especially to castration-resistant patients with activated STAT3, and preventing disease progression.

TLR3 Expression Induces Apoptosis in Human Non-Small-Cell Lung Cancer.

The prognostic value of Toll-like receptor 3 (TLR3) is debated in cancer, differing between tumor types, methods, and cell types. We recently showed for the first time that TLR3 expression on early stage non-small-cell lung cancer (NSCLC) results associated with a good prognosis. Here, we provide experimental evidences explaining the molecular reason behind TLR3's favorable prognostic role. We demonstrated that TLR3 activation in vitro induces apoptosis in lung cancer cell lines and, accordingly, that TLR3 expression is associated with caspase-3 activation in adenocarcinoma NSCLC specimens, both evaluated by immunohistochemistry. Moreover, we showed that TLR3 expression on cancer cells contributes to activate the CD103+ lung dendritic cell subset, that is specifically associated with processing of antigens derived from apoptotic cells and their presentation to CD8+ T lymphocytes. These findings point to the relevant role of TLR3 expression on lung cancer cells and support the use of TLR3 agonists in NSCLC patients to re-activate local innate immune response.

The lung microbiota: role in maintaining pulmonary immune homeostasis and its implications in cancer development and therapy.

Like other body districts, lungs present a complex bacteria community. An emerging function of lung microbiota is to promote and maintain a state of immune tolerance, to prevent uncontrolled and not desirable inflammatory response caused by inhalation of harmless environmental stimuli. This effect is mediated by a continuous dialog between commensal bacteria and immune cells resident in lungs, which express a repertoire of sensors able to detect microorganisms. The same receptors are also involved in the recognition of pathogens and in mounting a proper immune response. Due to its important role in preserving lung homeostasis, the lung microbiota can be also considered a mirror of lung health status. Indeed, several studies indicate that lung bacterial composition drastically changes during the occurrence of pulmonary pathologies, such as lung cancer, and the available data suggest that the modifications of lung microbiota can be part of the etiology of tumors in lungs and can influence their progression and response to therapy. These results provide the scientific rationale to analyze lung microbiota composition as biomarker for lung cancer and to consider lung microbiota a new potential target for therapeutic intervention to reprogram the antitumor immune microenvironment. In the present review, we discussed about the role of lung microbiota in lung physiology and summarized the most relevant data about the relationship between lung microbiota and cancer.

Toll-like receptor 3 as a new marker to detect high risk early stage Non-Small-Cell Lung Cancer patients.

Immune and epithelial cells express TLR3, a receptor deputed to respond to microbial signals activating the immune response. The prognostic value of TLR3 in cancer is debated and no data are currently available in NSCLC, for which therapeutic approaches that target the immune system are providing encouraging results. Dissecting the lung immune microenvironment could provide new prognostic markers, especially for early stage NSCLC for which surgery is the only treatment option. In this study we investigated the expression and the prognostic value of TLR3 on both tumor and immune compartments of stage I NSCLCs. In a cohort of 194 NSCLC stage I, TLR3 immunohistochemistry expression on tumor cells predicted a favorable outcome of early stage NSCLC, whereas on the immune cells infiltrating the tumor stroma, TLR3 expression associated with a poor overall survival. Patients with TLR3-positive immune infiltrating cells, but not tumor cells showed a worse prognosis compared with all other patients. The majority of TLR3-expressing immune cells resulted to be macrophages and TLR3 expression associates with PD-1 expression. TLR3 has an opposite prognostic significance when expressed on tumor or immune cells in early stage NCSCL. Analysis of TLR3 in tumor and immune cells can help in identifying high risk stage I patients for which adjuvant treatment would be beneficial.

Inhibition of DNA Repair Mechanisms and Induction of Apoptosis in Triple Negative Breast Cancer Cells Expressing the Human Herpesvirus 6 U94.

Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancers. In spite of initial good response to chemotherapy, the prognosis of TNBC remains poor and no effective specific targeted therapy is readily available. Recently, we demonstrated the ability of U94, the latency gene of human herpes virus 6 (HHV-6), to interfere with proliferation and with crucial steps of the metastatic cascade by using MDA-MB 231 TNBC breast cancer cell line. U94 expression was also associated with a partial mesenchymal-to-epithelial transition (MET) of cells, which displayed a less aggressive phenotype. In this study, we show the ability of U94 to exert its anticancer activity on three different TNBC cell lines by inhibiting DNA damage repair genes, cell cycle and eventually leading to cell death following activation of the intrinsic apoptotic pathway. Interestingly, we found that U94 acted synergistically with DNA-damaging drugs. Overall, we provide evidence that U94 is able to combat tumor cells with different mechanisms, thus attesting for the great potential of this molecule as a multi-target drug in cancer therapy.

Local Administration of Caloric Restriction Mimetics to Promote the Immune Control of Lung Metastases.

Caloric restriction mimetics (CRMs), compounds that mimic the biochemical effects of nutrient deprivation, administered via systemic route promote antitumor effects through the induction of autophagy and the modulation of the immune microenvironment; however, collateral effects due to metabolic changes and the possible weight loss might potentially limit their administration at long term. Here, we investigated in mice local administration of CRMs via aerosol to reduce metastasis implantation in the lung, whose physiologic immunosuppressive status favors tumor growth. Hydroxycitrate, spermidine, and alpha-lipoic acid, CRMs that target different metabolic enzymes, administered by aerosol, strongly reduced implantation of intravenously injected B16 melanoma cells without overt signs of toxicity, such as weight loss and changes in lung structure. Cytofluorimetric analysis of lung immune infiltrates revealed a significant increase of alveolar macrophages and CD103+ dendritic cells in mice treated with CRMs that paralleled an increased recruitment and activation of both CD3 T lymphocytes and NK cells. These effects were associated with the upregulation of genes related to M1 phenotype, as IL-12 and STAT-1, and to the decrease of M2 genes, as IL-10 and STAT-6, in adherent fraction of lung immune infiltrate, as revealed by real-time PCR analysis. Thus, in this proof-of-principle study, we highlight the antitumor effect of CRM aerosol delivery as a new and noninvasive therapeutic approach to locally modulate immunosurveillance at the tumor site in the lung.

Modulation of peripheral blood immune cells by early use of steroids and its association with clinical outcomes in patients with metastatic non-small cell lung cancer treated with immune checkpoint inhibitors.

BACKGROUND: Steroids are frequently used in patients with metastatic non-small cell lung cancer (mNSCLC), but they could be detrimental for patients treated with immune checkpoint inhibitors (ICIs). Here, we assessed the association between early use of steroids, clinical outcomes and peripheral immune blood cells modulation in patients with mNSCLC treated with ICIs. METHODS: We reviewed patients with mNSCLC treated at our institution between April 2013 and December 2017. Early use of steroids was defined as the use of a daily prednisone-equivalent dose ≥10 mg for at least 1 day within 28 days after ICI initiation. Peripheral immune blood cell counts were retrieved at baseline and at 4 and 6 weeks after ICI initiation. RESULTS: Out of 151 patients included, 35 (23%) made early use of steroids that was associated with poor disease control (OR 0.32, p=0.006), progression-free survival (HR 1.80, p=0.003) and overall survival (HR 2.60, p<0.001). Early use of steroids significantly correlated with higher median absolute neutrophil count, neutrophil to lymphocyte ratio (NLR) and derived NLR, and lower median absolute and relative eosinophil count, both at 4 and 6 weeks after ICI initiation. CONCLUSIONS: In patients with mNSCLC treated with ICIs, early use of steroids was associated with worse clinical outcomes and remarkable modulation of peripheral blood immune cells, which could contribute to restraining the activation of antitumour immunity. If confirmed in prospective studies, these findings would highlight the importance of carefully evaluating and, whenever possible, avoiding steroids during early phases of ICI treatment.

Antibody-Fc/FcR Interaction on Macrophages as a Mechanism for Hyperprogressive Disease in Non-small Cell Lung Cancer Subsequent to PD-1/PD-L1 Blockade.

PURPOSE: Hyperprogression (HP), a paradoxical boost in tumor growth, was described in a subset of patients treated with immune checkpoint inhibitors (ICI). Neither clinicopathologic features nor biological mechanisms associated with HP have been identified. EXPERIMENTAL DESIGN: Among 187 patients with non-small cell lung cancer (NSCLC) treated with ICI at our institute, cases with HP were identified according to clinical and radiologic criteria. Baseline histologic samples from patients treated with ICI were evaluated by IHC for myeloid and lymphoid markers. T-cell-deficient mice, injected with human lung cancer cells and patient-derived xenografts (PDX) belonging to specific mutational subsets, were assessed for tumor growth after treatment with antibodies against mouse and human programmed death receptor-1 (PD-1). The immune microenvironment was evaluated by flow cytometry and IHC. RESULTS: Among 187 patients, 152 were evaluable for clinical response. We identified four categories: 32 cases were defined as responders (21%), 42 patients with stable disease (27.7%), 39 cases were defined as progressors (25.7%), and 39 patients with HP (25.7%). Pretreatment tissue samples from all patients with HP showed tumor infiltration by M2-like CD163+CD33+PD-L1+ clustered epithelioid macrophages. Enrichment by tumor-associated macrophages (TAM) was observed, even in tumor nodules from immunodeficient mice injected with human lung cancer cells and with PDXs. In these models, tumor growth was enhanced by treatment with anti-PD-1 but not anti-PD-1 F(ab)2 fragments. CONCLUSIONS: These results suggest a crucial role of TAM reprogramming, upon Fc receptor engagement by ICI, eventually inducing HP and provide clues on a distinctive immunophenotype potentially able to predict HP.See related commentary by Knorr and Ravetch, p. 904.

HER2 signaling regulates the tumor immune microenvironment and trastuzumab efficacy.

Through whole-transcriptome profiling of HER2+ breast carcinomas (BCs), we previously showed that those sensitive to trastuzumab are addicted to this oncoprotein and are enriched in immune pathways, raising the hypothesis that HER2 itself regulates immune cell recruitment. In the present study we investigated the relationship between HER2 activity and the pro-trastuzumab tumor immune milieu. Gene expression profiling and immunohistochemistry analysis of 53 HER2+ BCs showed that trastuzumab-sensitive tumors expressed significantly higher levels of chemokines involved in immune cell recruitment, with higher infiltration of T cells and monocytes, and higher levels of PD-1 ligands than tumors that do not benefit from trastuzumab. In vitro analysis in HER2+ BC cells revealed that CCL2 production was induced by HER2 stimulation with EGF/HRG via the PI3K-NF-kB axis, and down-modulated by HER2 inhibition with trastuzumab. CCL2 expression was higher in HER2+/ER- than HER2+/ER+ BC cell lines, and degradation of ER by fulvestrant induced an enhancement in NF-κB transcriptional activity and consequent CCL2 expression. Trastuzumab efficacy relied on CCL2 levels and monocytes present in the tumor microenvironment in FVB mice bearing HER2+ mammary carcinoma cells. HER2 signals were also found to sustain the expression of PD-1 ligands in tumor cells via the MEK pathway. Overall, our results support the concept that the activated HER2 oncogene regulates recruitment and activation of tumor infiltrating immune cells and trastuzumab activity by inducing CCL2 and PD-1 ligands and that ER activity negatively controls the HER2-driven pro-trastuzumab tumor microenvironment.

Modulation of Pulmonary Microbiota by Antibiotic or Probiotic Aerosol Therapy: A Strategy to Promote Immunosurveillance against Lung Metastases.

Pulmonary immunological tolerance to inhaled particulates might create a permissive milieu for lung metastasis. Lung microbiota contribute to pulmonary tolerance; here, we explored whether its manipulation via antibiotic or probiotic aerosolization favors immune response against melanoma metastasis. In lungs of vancomycin/neomycin-aerosolized mice, a decrease in bacterial load was associated with reduced regulatory T cells and enhanced T cell and NK cell activation that paralleled a significant reduction of melanoma B16 lung metastases. Reduction of metastases also occurred in lungs transplanted with bacterial isolates from antibiotic-treated lungs. Aerosolized Lactobacillus rhamnosus strongly promoted immunity against B16 lung metastases as well. Furthermore, probiotics or antibiotics improved chemotherapy activity against advanced B16 metastases. Thus, we identify a role for lung microbiota in metastasis and show that its targeting via aerosolization is a therapy that can prevent metastases and enhance responses to chemotherapy.

Exploiting poly(I:C) to induce cancer cell apoptosis.

TLR3 belong to the Toll-like receptors family, it is mainly expressed on immune cells where it senses pathogen-associated molecular patterns and initiates innate immune response. TLR3 agonist poly(I:C) was developed to mimic pathogens infection and boost immune system activation to promote anti-cancer therapy. Accordingly, TLR agonists were included in the National Cancer Institute list of immunotherapeutic agents with the highest potential to cure cancer. Besides well known effects on immune cells, poly(I:C) was also shown, in experimental models, to directly induce apoptosis in cancer cells expressing TLR3. This review presents the current knowledge on the mechanism of poly(I:C)-induced apoptosis in cancer cells. Experimental evidences on positive or negative regulators of TLR3-mediated apoptosis induced by poly(I:C) are reported and strategies are proposed to successfully promote this event in cancer cells. Cancer cells apoptosis is an additional arm offered by poly(I:C), besides activation of immune system, for the treatment of various type of cancer. A further dissection of TLR3 signaling would contribute to greater resolution of the critical steps that impede full exploitation of the poly(I:C)-induced apoptosis. Experimental evidences about negative regulator of poly(I:C)-induced apoptotic program should be considered in combinations with TLR3 agonists in clinical trials.

Activation of NK cell cytotoxicity by aerosolized CpG-ODN/poly(I:C) against lung melanoma metastases is mediated by alveolar macrophages.

Controversies remain about NK cells direct responsiveness to Toll-like receptor (TLR) agonists or dependence on macrophages. In a melanoma lung metastasis model, aerosolized TLR9 and TLR3 agonists have been reported to induce antitumor immunity through NK cells activation. In the current study, we demonstrated that in vitro TLR9/TLR3 stimulation induced IFN-γ secretion by NK cells, but an increase in their cytotoxicity was detected only after NK cells co-culture with in vitro TLR9/TLR3 agonists pretreated alveolar macrophages. Alveolar macrophages from melanoma lung metastases-bearing mice, treated with aerosolized TLR agonists, also promoted NK cell cytotoxicity. Activated NK cells from lungs of melanoma metastases-bearing mice that were given aerosolized TLR9/TLR3 agonists were able to polarize naive alveolar macrophages toward a M1-like phenotype. Our results demonstrate that activation of NK cells in the lung after TLR engagement is mediated by alveolar macrophages and that activated NK cells shape macrophage behavior.

Reprogramming the lung microenvironment by inhaled immunotherapy fosters immune destruction of tumor.

Due to their constant exposure to inhaled antigens, lungs represent a particularly immunosuppressive environment that limits excessive immune responses; however, cancer cells can exploit this unique environment for their growth. We previously described the ability of aerosolized CpG-ODN combined with Poly(I:C) (TLR9 and TLR3 agonists, respectively) to promote antitumor immunity in a B16 melanoma lung metastasis model. Here, we explored the possibility of improving the therapeutic efficacy of TLR9/TLR3 agonist combinations by including in the inhalant either an antibody directed to both Ly6G and Ly6C markers to locally deplete myeloid-derived suppressive cells (MDSCs) or IFNα to directly activate the natural killer (NK) and macrophage innate immune cells in the lung. Addition of nebulized anti-MDSC antibody RB6-8C5 to aerosolized CpG-ODN/Poly(I:C) resulted in reduced mRNA levels of immunsuppressive molecules (IL10, Arg-1, and Nos2), increased activation of resident NK cells and improved treatment outcome, with a significant reduction in established B16 melanoma lung metastases compared to treatment with CpG-ODN/Poly(I:C) alone. Likewise, addition of aerosolized IFNα led to increased mRNA levels of proinflammatory cytokines (IL15 and IFNγ) in the lung and recruitment of highly activated NK cells, with no evident signs of toxicity and with a significantly improved antitumor effect as compared with aerosolized CpG-ODN/Poly(I:C). Combining both IFNα and RB6-8C5 with CpG-ODN/Poly(I:C) did not produce an additive effect compared to IFNα + CpG-ODN/Poly(I:C) or RB6-8C5 + CpG-ODN/Poly(I:C). Our results indicate that the inhalation therapy is a feasible and non-invasive strategy to deliver immunodulatory molecules, including antibodies and cytokines that reprogram the lung tumor microenvironment to foster immune destruction of tumors.

Expression and prognostic significance of the autoimmune regulator gene in breast cancer cells.

The autoimmune regulator gene (AIRE) plays a fundamental role in tolerance by promoting the expression of tissue-specific antigens in medullary thymic epithelial cells (mTECs). Recently, AIRE expression was detected also in human keratinocytes and in tumors originating in stratified epithelia. Here, we tested whether AIRE is expressed in cancer cells. We analyzed AIRE expression in cancer cases from The Cancer Genome Atlas (TCGA) RNA-seq dataset and we found association with better outcome. AIRE protein expression was verified by immunohistochemistry in a cohort of 39 human breast cancer specimens and its prognostic relevance was confirmed in microarray-based gene expression data set NKI-295 and KM-Plotter. Both in the RNA-seq and gene expression datasets analyzed, AIRE expression was an independent strong prognostic factor for relapse-free survival (RFS), particularly in estrogen receptor-positive tumors. Enrichment of translation-related pathways was observed in AIRE-expressing tumors by Ingenuity Pathway Analysis and a significant increase of cells in G1 phase and activation of caspase cascades was induced by AIRE transfection in breast cancer luminal cell lines, suggesting that AIRE-induced over-translation of proteins lead to cycle arrest and apoptosis. These data are the first to identify AIRE expression in breast cancer and an association with prognosis.

CpG-oligodeoxynucleotides exert remarkable antitumor activity against diffuse malignant peritoneal mesothelioma orthotopic xenografts.

BACKGROUND: Diffuse malignant peritoneal mesothelioma (DMPM) is a rare and locally aggressive disease. DMPM prognosis is dismal, mainly due to the lack of effective treatment options and the development of new therapeutic strategies is urgently needed. In this context, novel immunotherapy approaches can be explored in an attempt to improve DMPM patients' survival. METHODS: We tested the efficacy of CpG-oligodeoxynucleotides (CpG-ODN), synthetic DNA sequences recognized by Toll-like receptor 9 and able to induce innate/adaptive immune response, in two DMPM orthotopic xenografts (MesoII and STO), which properly recapitulate the dissemination pattern of the disease in the peritoneal cavity. Severe combined immunodeficiency mice carrying DMPM xenografts were treated at different stages of tumor development with i.p. delivered CpG-ODN1826 for 4 weeks. CpG-ODN1826-induced modulation in the composition of peritoneal immune infiltrate was assessed by flow cytometry. RESULTS: When administered to early-stage tumors (i.e., 4 days after i.p. DMPM cell injection in mice), the agent exhibited impressive efficacy against MesoII by completely inhibiting tumor take and ascites development (no evidence of tumor masses and ascites in 6/6 mice at necropsy), and also impaired STO tumor take and growth (4/6 tumor-free mice; i.p. tumor masses reduced by 94 % in the 2 remaining mice, P = 0.00005). Interestingly, when tested against late-stage STO tumors (i.e., 11 days after i.p. DMPM cell injection in mice), CpG-ODN1826 was still able to reduce the growth of i.p. tumor masses by 66 % (P = 0.0009). Peritoneal washings of tumor-bearing mice revealed a strong increase of macrophage infiltration together with a decrease in the presence of B-1 cells and a reduced IgM concentration after CpG-ODN1826 treatment. CONCLUSIONS: Our results indicate that locally administered CpG-ODN1826 is able to markedly affect the growth of both early- and late-stage DMPM orthotopic xenografts in the absence of severe side effects, and suggest a possible clinical role for the agent in the therapy of DMPM.

Taxanes enhance trastuzumab-mediated ADCC on tumor cells through NKG2D-mediated NK cell recognition.

Recent clinical data indicate a synergistic therapeutic effect between trastuzumab and taxanes in neoadjuvantly treated HER2-positive breast cancer (BC) patients. In HER2+ BC experimental models and patients, we investigated whether this synergy depends on the ability of drug-induced stress to improve NK cell effectiveness and thus trastuzumab-mediated ADCC. HER2+ BC cell lines BT474 and MDAMB361 treated with docetaxel showed up-modulation of NK activator ligands both in vitro and in vivo, accompanied by a 15-40% increase in in vitro trastuzumab-mediated ADCC; antibodies blocking the NKG2D receptor significantly reduced this enhancement. NKG2D receptor expression was increased by docetaxel treatment in circulating and splenic NK cells from mice xenografted with tumor cells, an increase related to expansion of the CD11b+Ly6G+ cell population. Accordingly, NK cells derived from HER2+ BC patients after treatment with taxane-containing therapy expressed higher levels of NKG2D receptor than before treatment. Moreover, plasma obtained from these patients recapitulated the modulation of NKG2D on healthy donors' NK cells, improving their trastuzumab-mediated activity in vitro. This enhancement occurred mainly using plasma from patients with low NKG2D basal expression. Our results indicate that taxanes increase tumor susceptibility to ADCC by acting on tumor and NK cells, and suggest that taxanes concomitantly administered with trastuzumab could maximize the antibody effect, especially in patients with low basal immune effector cytotoxic activity.