In Vitro SARS-CoV-2 Infection of Microvascular Endothelial Cells: Effect on Pro-Inflammatory Cytokine and Chemokine Release.

In the novel pandemic of Coronavirus Disease 2019, high levels of pro-inflammatory cytokines lead to endothelial activation and dysfunction, promoting a pro-coagulative state, thrombotic events, and microvasculature injuries. The aim of the present work was to investigate the effect of SARS-CoV-2 on pro-inflammatory cytokines, tissue factor, and chemokine release, with Human Microvascular Endothelial Cells (HMEC-1). ACE2 receptor expression was evaluated by western blot analysis. SARS-CoV-2 infection was assessed by one-step RT-PCR until 7 days post-infection (p.i.), and by Transmission Electron Microscopy (TEM). IL-6, TNF-α, IL-8, IFN-α, and hTF mRNA expression levels were detected by RT-PCR, while cytokine release was evaluated by ELISA. HMEC-1 expressed ACE2 receptor and SARS-CoV-2 infection showed a constant viral load. TEM analysis showed virions localized in the cytoplasm. Expression of IL-6 at 24 h and IFN-α mRNA at 24 h and 48 h p.i. was higher in infected than uninfected HMEC-1 (p < 0.05). IL-6 levels were significantly higher in supernatants from infected HMEC-1 (p < 0.001) at 24 h, 48 h, and 72 h p.i., while IL-8 levels were significantly lower at 24 h p.i. (p < 0.001). These data indicate that in vitro microvascular endothelial cells are susceptible to SARS-CoV-2 infection but slightly contribute to viral amplification. However, SARS-CoV-2 infection might trigger the increase of pro-inflammatory mediators.

Characterisation of Progressive Skeletal Muscle Fibrosis in the Mdx Mouse Model of Duchenne Muscular Dystrophy: An In Vivo and In Vitro Study.

Duchenne muscular dystrophy (DMD) is a rare genetic disease leading to progressive muscle wasting, respiratory failure, and cardiomyopathy. Although muscle fibrosis represents a DMD hallmark, the organisation of the extracellular matrix and the molecular changes in its turnover are still not fully understood. To define the architectural changes over time in muscle fibrosis, we used an mdx mouse model of DMD and analysed collagen and glycosaminoglycans/proteoglycans content in skeletal muscle sections at different time points during disease progression and in comparison with age-matched controls. Collagen significantly increased particularly in the diaphragm, quadriceps, and gastrocnemius in adult mdx, with fibrosis significantly correlating with muscle degeneration. We also analysed collagen turnover pathways underlying fibrosis development in cultured primary quadriceps-derived fibroblasts. Collagen secretion and matrix metalloproteinases (MMPs) remained unaffected in both young and adult mdx compared to wt fibroblasts, whereas collagen cross-linking and tissue inhibitors of MMP (TIMP) expression significantly increased. We conclude that, in the DMD model we used, fibrosis mostly affects diaphragm and quadriceps with a higher collagen cross-linking and inhibition of MMPs that contribute differently to progressive collagen accumulation during fibrotic remodelling. This study offers a comprehensive histological and molecular characterisation of DMD-associated muscle fibrosis; it may thus provide new targets for tailored therapeutic interventions.

The Educational Program of Macrophages toward a Hyperprogressive Disease-Related Phenotype Is Orchestrated by Tumor-Derived Extracellular Vesicles.

Hyperprogressive disease (HPD), an aggressive acceleration of tumor growth, was observed in a group of cancer patients treated with anti-PD1/PDL1 antibodies. The presence of a peculiar macrophage subset in the tumor microenvironment is reported to be a sort of "immunological prerequisite" for HPD development. These macrophages possess a unique phenotype that it is not clear how they acquire. We hypothesized that certain malignant cells may promote the induction of an "HPD-related" phenotype in macrophages. Bone-marrow-derived macrophages were exposed to the conditioned medium of five non-small cell lung cancer cell lines. Macrophage phenotype was analyzed by microarray gene expression profile and real-time PCR. We found that human NSCLC cell lines, reported as undergoing HPD-like tumor growth in immunodeficient mice, polarized macrophages towards a peculiar pro-inflammatory phenotype sharing both M1 and M2 features. Lipid-based factors contained in cancer cell-conditioned medium induced the over-expression of several pro-inflammatory cytokines and the activation of innate immune receptor signaling pathways. We also determined that tumor-derived Extracellular Vesicles represent the main components involved in the observed macrophage re-education program. The present study might represent the starting point for the future development of diagnostic tools to identify potential hyperprogressors.

Live or Heat-Killed Lactobacillus rhamnosus Aerosolization Decreases Adenomatous Lung Cancer Development in a Mouse Carcinogen-Induced Tumor Model.

An immunosuppressive microenvironment in lung concurs to pre-malignant lesions progression to cancer. Here, we explore if perturbing lung microbiota, which contribute to immunosuppression, by antibiotics or probiotic aerosol interferes with lung cancer development in a mouse carcinogen-induced tumor model. Urethane-injected mice were vancomycin/neomycin (V/N)-aerosolized or live or dead L. rhamnosus GG (L.RGG)-aerosolized, and tumor development was evaluated. Transcriptional profiling of lungs and IHC were performed. Tumor nodules number, diameter and area were reduced by live or heat-killed L.RGG, while only a decrease in nodule diameter was observed in V/N-treated lungs. Both L.RGG and V/N reduced Tregs in the lung. In L.RGG-treated groups, the gene encoding the joining chain (J chain) of immunoglobulins was increased, and higher J chain protein and IgA levels were observed. An increased infiltration of B, NK and myeloid-derived cells was predicted by TIMER 2.0. The Kaplan-Meier plotter revealed an association between high levels of J chain mRNA and good prognosis in lung adenocarcinoma patients that correlated with increased B and CD4 T cells and reduced Tregs and M2 macrophages. This study highlights L.RGG aerosol efficacy in impairing lung cancer growth by promoting local immunity and points to this non-invasive strategy to treat individuals at risk of lung cancer.

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.

Three-Dimensional Cell Cultures as an In Vitro Tool for Prostate Cancer Modeling and Drug Discovery.

In the last decade, three-dimensional (3D) cell culture technology has gained a lot of interest due to its ability to better recapitulate the in vivo organization and microenvironment of in vitro cultured cancer cells. In particular, 3D tumor models have demonstrated several different characteristics compared with traditional two-dimensional (2D) cultures and have provided an interesting link between the latter and animal experiments. Indeed, 3D cell cultures represent a useful platform for the identification of the biological features of cancer cells as well as for the screening of novel antitumor agents. The present review is aimed at summarizing the most common 3D cell culture methods and applications, with a focus on prostate cancer modeling and drug discovery.

Generation of clinical-grade CD19-specific CAR-modified CD8+ memory stem cells for the treatment of human B-cell malignancies.

Long-lived, self-renewing, multipotent T memory stem cells (TSCM) can trigger profound and sustained tumor regression but their rareness poses a major hurdle to their clinical application. Presently, clinically compliant procedures to generate relevant numbers of this T-cell population are undefined. Here, we provide a strategy for deriving large numbers of clinical-grade tumor-redirected TSCM starting from naive precursors. CD8(+)CD62L(+)CD45RA(+) naive T cells enriched by streptamer-based serial-positive selection were activated by CD3/CD28 engagement in the presence of interleukin-7 (IL-7), IL-21, and the glycogen synthase-3ß inhibitor TWS119, and genetically engineered to express a CD19-specific chimeric antigen receptor (CD19-CAR). These conditions enabled the generation of CD19-CAR-modified CD8(+) TSCM that were phenotypically, functionally, and transcriptomically equivalent to their naturally occurring counterpart. Compared with CD8(+) T cells generated with clinical protocols currently under investigation, CD19-CAR-modified CD8(+) TSCM exhibited enhanced metabolic fitness and mediated robust, long-lasting antitumor responses against systemic acute lymphoblastic leukemia xenografts. This clinical-grade platform provides the basis for a phase 1 trial evaluating the activity of CD19-CAR-modified CD8(+) TSCM in patients with B-cell malignancies refractory to prior allogeneic hematopoietic stem cell transplantation.

3D-spheroids: What can they tell us about pancreatic ductal adenocarcinoma cell phenotype?

We aimed at analyzing the effect of the 3D-arrangement on the expression of some genes and proteins which play a key role in pancreatic adenocarcinoma (PDAC) progression in HPAF-II, HPAC and PL45 PDAC cells cultured in either 2D-monolayers or 3D-spheroids. Cytokeratins 7, 8, 18, 19 were differently expressed in 3D-spheroids compared to 2D-monolayers. Syndecan 1 was upregulated in HPAF-II and PL45 3D-spheroids, and downregulated in HPAC. Heparanase mRNA levels were almost unchanged in HPAF-II, and increased in HPAC and PL45 3D-spheroids. Hyaluronan synthase (HAS) 2 and 3 mRNA increased in all 3D-spheroids compared to 2D-monolayers. CD44 and CD44s were expressed to a lower extent in HPAF-II and HPAC 3D-spheroids. By contrast, the CD44s/v3 and the CD44s/v6 ratio increased in HPAC and PL45 3D-spheroids, compared to 2D-monolayers. The expression of MMP-7 was strongly upregulated in 3D-spheroids. STAT3 was similarly expressed 3D-spheroids or 2D-monolayers, while pSTAT3 was almost undetectable in 2D-monolayers and strongly upregulated in 3D-spheroids. These results suggest that 3D-spheroids represent a cell culture model that allows the characterization of PDAC cell phenotype, adding new information that contributes to a better understanding of the biology and behavior of PDAC cells.

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.

Inherent transcriptional signatures of NK cells are associated with response to IFNα + rivabirin therapy in patients with Hepatitis C Virus.

BACKGROUND: Differences in the expression of Natural Killer cell receptors have been reported to reflect divergent clinical courses in patients with chronic infections or tumors. However, extensive molecular characterization at the transcriptional level to support this view is lacking. The aim of this work was to characterize baseline differences in purified NK cell transcriptional activity stratified by response to treatment with PEG-IFNα/RBV in patients chronically infected with HCV. METHODS: To this end we here studied by flow cytometer and gene expression profile, phenotypic and transcriptional characteristics of purified NK cells in patients chronically infected with HCV genotype-1 virus who were subsequently treated with PEG-IFNα/RBV. Results were further correlated with divergent clinical response obtained after treatment. RESULTS: The pre-treatment transcriptional patterns of purified NK cells from patients subsequently undergoing a sustained virologic response (SVR) clearly segregated from those of non-responder (NR) patients. A set of 476 transcripts, including molecules involved in RNA processing, ubiquitination pathways as well as HLA class II signalling were differently expressed among divergent patients. In addition, treatment outcome was associated with differences in surface expression of NKp30 and NKG2D. A complex relationship was observed that suggested for extensive post-transcriptional editing. Only a small number of the NK cell transcripts identified were correlated with chronic HCV infection/replication indicating that inherent transcriptional activity prevails over environment effects such as viral infection. CONCLUSIONS: Collectively, inherent/genetic modulation of NK cell transcription is involved in setting the path to divergent treatment outcomes and could become useful to therapeutic advantage.

Sodium glucose cotransporter 1 ligand BLF501 as a novel tool for management of gastrointestinal mucositis.

BACKGROUND: Recent studies demonstrated that engagement of sodium glucose transporter 1 (SGLT-1) by orally administered D-glucose protects the intestinal mucosa from lipopolysaccharide (LPS)-induced injury. We tested whether SGLT-1 engagement might protect the intestinal mucosa from doxorubicin (DXR)- and 5-fluorouracil (5-FU)-induced injury in animal models mimicking acute or chronic mucositis. METHODS: Mice were treated intraperitoneally with DXR, alone or in combination with 5-FU, and orally with BLF501, a glucose-derived synthetic compound with high affinity for SGLT-1. Intestinal mucosal epithelium integrity was assessed by histological analysis, cellular proliferation assays, real-time PCR gene expression assays and Western blot assays. Student's t-test (paired two-tailed) and χ2 analyses were used for comparisons between groups. Differences were considered significant at p < 0.05. RESULTS: BLF501 administration in mice treated with DXR and/or 5-FU decreased the injuries to the mucosa in terms of epithelial integrity and cellular proliferative ability. Co-treatment with BLF501 led to a normal expression and distribution of both zonula occludens-1 (ZO-1) and beta-catenin, which were underexpressed after treatment with either chemotherapeutic agent alone. BLF501 administration also restored normal expression of caspase-3 and ezrin/radixin/moesin (ERM), which were overexpressed after treatment with DXR and 5-FU. In SGLT1-/- mice, BLF501 had no detectable effects. BLF501 administration in wild-type mice with growing A431 tumors did not modify antitumor activity of DXR. CONCLUSIONS: BLF501-induced protection of the intestinal mucosa is a promising novel therapeutic approach to reducing the severity of chemotherapy-induced mucositis.

Differentiation States of Phenotypic Transition of Melanoma Cells Are Revealed by 3D Cell Cultures.

Melanoma is characterized by high metastatic potential favored by the epithelial-to-mesenchymal transition (EMT), leading melanoma cells to exhibit a spectrum of typical EMT markers. This study aimed to analyze the expression of EMT markers in A375 and BLM melanoma cell lines cultured in 2D monolayers and 3D spheroids using morphological and molecular methods. The expression of EMT markers was strongly affected by 3D arrangement and revealed a hybrid phenotype for the two cell lines. Indeed, although E-cadherin was almost undetectable in both A375 and BLM cells, cortical actin was detected in A375 2D monolayers and 3D spheroids and was strongly expressed in BLM 3D spheroids. The mesenchymal marker N-cadherin was significantly up-regulated in A375 3D spheroids while undetectable in BLM cells, but vimentin was similarly expressed in both cell lines at the gene and protein levels. This pattern suggests that A375 cells exhibit a more undifferentiated/mesenchymal phenotype, while BLM cells have more melanocytic/differentiated characteristics. Accordingly, the Zeb1 and 2, Slug, Snail and Twist gene expression analyses showed that they were differentially expressed in 2D monolayers compared to 3D spheroids, supporting this view. Furthermore, A375 cells are characterized by a greater invasive potential, strongly influenced by 3D arrangement, compared to the BLM cell line, as evaluated by SDS-zymography and TIMPs gene expression analysis. Finally, TGF-ß1, a master controller of EMT, and lysyl oxidase (LOX), involved in melanoma progression, were strongly up-regulated by 3D arrangement in the metastatic BLM cells alone, likely playing a role in the metastatic phases of melanoma progression. Overall, these findings suggest that A375 and BLM cells possess a hybrid/intermediate phenotype in relation to the expression of EMT markers. The former is characterized by a more mesenchymal/undifferentiated phenotype, while the latter shows a more melanocytic/differentiated phenotype. Our results contribute to the characterization of the role of EMT in melanoma cells and confirm that a 3D cell culture model could provide deeper insight into our understanding of the biology of melanoma.

Impact of in vitro SARS-CoV-2 infection on breast cancer cells.

The pandemic of coronavirus disease 19 (COVID-19), caused by severe respiratory syndrome coronavirus 2 (SARS-CoV-2), had severe repercussions for breast cancer patients. Increasing evidence indicates that SARS-CoV-2 infection may directly impact breast cancer biology, but the effects of SARS-CoV-2 on breast tumor cells are still unknown. Here, we analyzed the molecular events occurring in the MCF7, MDA-MB-231 and HCC1937 breast cancer cell lines, representative of the luminal A, basal B/claudin-low and basal A subtypes, respectively, upon SARS-CoV-2 infection. Viral replication was monitored over time, and gene expression profiling was conducted. We found that MCF7 cells were the most permissive to viral replication. Treatment of MCF7 cells with Tamoxifen reduced the SARS-CoV-2 replication rate, suggesting an involvement of the estrogen receptor in sustaining virus replication in malignant cells. Interestingly, a metagene signature based on genes upregulated by SARS-CoV-2 infection in all three cell lines distinguished a subgroup of premenopausal luminal A breast cancer patients with a poor prognosis. As SARS-CoV-2 still spreads among the population, it is essential to understand the impact of SARS-CoV-2 infection on breast cancer, particularly in premenopausal patients diagnosed with the luminal A subtype, and to assess the long-term impact of COVID-19 on breast cancer outcomes.

Cigarette smoke sustains immunosuppressive microenvironment inducing M2 macrophage polarization and viability in lung cancer settings.

BACKGROUND: It is amply demonstrated that cigarette smoke (CS) has a high impact on lung tumor progression worsening lung cancer patient prognosis and response to therapies. Alteration of immune cell types and functions in smokers' lungs have been strictly related with smoke detrimental effects. However, the role of CS in dictating an inflammatory or immunosuppressive lung microenvironment still needs to be elucidated. Here, we investigated the effect of in vitro exposure to cigarette smoke extract (CSE) focusing on macrophages. METHODS: Immortalized murine macrophages RAW 264.7 cells were cultured in the presence of CS extract and their polarization has been assessed by Real-time PCR and cytofluorimetric analysis, viability has been assessed by SRB assay and 3D-cultures and activation by exposure to Poly(I:C). Moreover, interaction with Lewis lung carcinoma (LLC1) murine cell models in the presence of CS extract were analyzed by confocal microscopy. RESULTS: Obtained results indicate that CS induces macrophages polarization towards the M2 phenotype and M2-phenotype macrophages are resistant to the CS toxic activity. Moreover, CS impairs TLR3-mediated M2-M1 phenotype shift thus contributing to the M2 enrichment in lung smokers. CONCLUSIONS: These findings indicate that, in lung cancer microenvironment of smokers, CS can contribute to the M2-phenotype macrophages prevalence by different mechanisms, ultimately, driving an anti-inflammatory, likely immunosuppressive, microenvironment in lung cancer smokers.

Influence of fatty acid-free diet on mammary tumor development and growth rate in HER-2/Neu transgenic mice.

Numerous investigations have found a relationship between higher risk of cancer and increased intake of fats, while results of clinical studies of fat reduction and breast cancer recurrence have been mixed. A diet completely free of fats cannot be easily administered to humans, but experimental studies in mice can be done to determine whether this extreme condition influences tumor development. Here, we examined the effects of a FA-free diet on mammary tumor development and growth rate in female FVB-neu proto-oncogene transgenic mice that develop spontaneous multifocal mammary tumors after a long latency period. Mice were fed a fatty acid-free diet beginning at 112, 35, and 30 days of age. In all these experiments, tumor appearance was delayed, tumor incidence was reduced and the mean number of palpable mammary tumors per mouse was lower, as compared to standard diet-fed mice. By contrast, tumor growth rate was unaffected in mice fed the fatty acid-free diet. Plasma of mice fed the fatty acid-free diet revealed significantly higher contents of oleic, palmitoleic and 20:3ω9 acids and lower contents of linoleic and palmitic acids. In conclusion, these findings indicate that a FA-free diet reduces tumor incidence and latency but not tumor growth rate, suggesting that a reduction in dietary FAs in humans may have a protective effect on tumorigenesis but not on tumors once they appear.

Anti-tumor activity of CpG-ODN aerosol in mouse lung metastases.

Studies in preclinical models have demonstrated the superior anti-tumor effect of CpG oligodeoxynucleotides (CpG-ODN) when administered at the tumor site rather than systemically. We evaluated the effect of aerosolized CpG-ODN on lung metastases in mice injected with immunogenic N202.1A mammary carcinoma cells or weakly immunogenic B16 melanoma cells. Upon reaching the bronchoalveolar space, aerosolized CpG-ODN activated a local immune response, as indicated by production of IL-12p40, IFN-γ and IL-1ß and by recruitment and maturation of DC cells in bronchoalveolar lavage fluid of mice. Treatment with aerosolized CpG-ODN induced an expansion of CD4+ cells in lung and was more efficacious than systemic i.p. administration against experimental lung metastases of immunogenic N202.1A mammary carcinoma cells, whereas only i.p. delivery of CpG-ODN provided anti-tumor activity, which correlated with NK cell expansion in the lung, against lung metastases of the poorly immunogenic B16 melanoma. The inefficacy of aerosol therapy to induce NK expansion was related to the presence of immunosuppressive macrophages in B16 tumor-bearing lungs, as mice depleted of these cells by clodronate treatment responded to aerosol CpG-ODN through expansion of the NK cell population and significantly reduced numbers of lung metastases. Our results indicate that tumor immunogenicity and the tumor-induced immunosuppressive environment are critical factors to the success of CpG therapy in the lung, and point to the value of routine sampling of the lung immune environment in defining an optimal immunotherapeutic strategy.

High efficacy of CpG-ODN, cetuximab and cisplatin combination for very advanced ovarian xenograft tumors.

BACKGROUND: To mimic clinical treatment situations in advanced human ovarian disease, 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 responses, in combination with other possible therapeutic reagents in ovarian carcinoma ascites-bearing athymic mice. METHODS: Mice injected i.p. with IGROV-1 ovarian cancer cells were treated at different stages of ascites progression for 4 weeks with CpG-ODN, alone or in combination with Bevacizumab, Polyinosinic:Polycytidylic acid (Poly(I):Poly(C)), Gefitinib, Cetuximab and Cisplatin. Median survival time (MST) was calculated for each group. IGROV-1 cells treated or not with Cetuximab were assayed for antibody-dependent cellular cytotoxicity by 51Cr-release assay, and for macrophage antibody-dependent cell-mediated phagocytosis by flow cytometry. RESULTS: In mice treated when ascitic fluid began to accumulate, CpG-ODN combined with Bevacizumab, Poly(I):Poly(C) or Gefitinib did not significantly increase MST as compared with that using CpG-ODN alone, whereas MST in mice treated with CpG-ODN plus Cetuximab was significantly increased (>103 days for combination vs 62 days for CpG alone; P = 0.0008), with 4/8 mice alive at the end of the experiment. In experiments in mice showing increased abdominal volume and body weight (27.9 ± 0.8 g after vs 23 ± 1.1 g before tumor cell injection), treatment with Cisplatin in addition to CpG-ODN/Cetuximab led to significantly increased MST (105.5 days; P = 0.001), with all mice still alive at 85 days, over that using CpG-ODN/Cetuximab (66 days), Cetuximab/Cisplatin (18.5 days), Cisplatin (23 days) or saline (16 days). At a very advanced stage of disease (body weight: 31.4 ± 0.9 g), when more than half of control mice had to be sacrificed 6 days after starting treatments, the triple-combination therapy still increased MST (45 days; P = 0.0089) vs controls. CONCLUSIONS: CpG-ODN combination therapies that enhance the immune response in the tumor microenvironment and concomitantly target tumor cells are highly efficacious even in experimental advanced malignancies. Although differences in the distribution of TLR9 in mice and humans and the enrichment of this receptor on innate immune cells of athymic mice must be considered, our results indicate a promising strategy to treat ovarian cancer patients with bulky ascites.

The Emerging Role of the Microbiota in Breast Cancer Progression.

Emerging evidence suggests a profound association between the microbiota composition in the gastrointestinal tract and breast cancer progression. The gut microbiota plays a crucial role in modulating the immune response, releasing metabolites, and modulating estrogen levels, all of which have implications for breast cancer growth. However, recent research has unveiled a novel aspect of the relationship between the microbiota and breast cancer, focusing on microbes residing within the mammary tissue, which was once considered sterile. These localized microbial communities have been found to change in the presence of a tumor as compared to healthy mammary tissue, unraveling their potential contribution to tumor progression. Studies have identified specific bacterial species that are enriched within breast tumors and have highlighted the mechanisms by which even these microbes influence cancer progression through immune modulation, direct carcinogenic activity, and effects on cellular pathways involved in cell proliferation or apoptosis. This review aims to provide an overview of the current knowledge on the mechanisms of crosstalk between the gut/mammary microbiota and breast cancer. Understanding this intricate interplay holds promise for developing innovative therapeutic approaches.

Immunostaining patterns reveal potential morphogenetic role of Toll-like receptors 4 and 7 in the development of mouse respiratory system, liver and pancreas.

Toll-like receptors (TLRs) are the mammalian ortholog of Drosophila melanogaster protein Toll, originally identified for its involvement in embryonic development. In mammals, TLRs are mainly known for their ability to recognize pathogen- or damage-associated molecular patterns and, consequently, to initiate the immune response. However, it is becoming clear that TLRs can play a role also in mammal embryo development. We have previously described TLR4 and TLR7 expression in developing mouse peripheral nervous system and gastrointestinal tract. In the present study, we extended the investigation of TLR4 and TLR7 to the respiratory system and to the two main accessory organs of the digestive system, the liver and pancreas. TLR4 and TLR7 immunostaining was performed on mouse conceptuses collected at different stages, from E12 to E18. TLR4 and TLR7 immunoreactivity was evident in the embryo pancreas and liver at E12, while, in the respiratory apparatus, appeared at E14 and E17, respectively. Although further studies are required to elucidate the specific role of these TLRs in embryo development, the differential spatiotemporal TLR4 and TLR7 appearance may suggest that TLR expression in developing embryos is highly regulated for a possible their direct involvement in the formation of the organs and in the acquisition of immune-related features in preparation for the birth.

Reduction of Staphylococcus epidermidis in the mammary tumor microbiota induces antitumor immunity and decreases breast cancer aggressiveness.

The mammary gland hosts a microbiota, which differs between malignant versus normal tissue. We found that aerosolized antibiotics decrease murine mammary tumor growth and strongly limit lung metastasis. Oral absorbable antibiotics also reduced mammary tumors. In ampicillin-treated nodules, the immune microenvironment consisted of an M1 profile and improved T cell/macrophage infiltration. In these tumors, we noted an under-representation of microbial recognition and complement pathways, supported by TLR2/TLR7 protein and C3-fragment deposition reduction. By 16S rRNA gene profiling, we observed increased Staphylococcus levels in untreated tumors, among which we isolated Staphylococcus epidermidis, which had potent inflammatory activity and increased Tregs. Conversely, oral ampicillin lowered Staphylococcus epidermidis in mammary tumors and expanded bacteria promoting an M1 phenotype and reducing MDSCs and tumor growth. Ampicillin/paclitaxel combination improved the chemotherapeutic efficacy. Notably, an Amp-like signature, based on genes differentially expressed in murine tumors, identified breast cancer patients with better prognosis and high immune infiltration that correlated with a bacteria response signature. This study highlights the significant influence of mammary tumor microbiota on local immune status and the relevance of its treatment with antibiotics, in combination with breast cancer therapies.