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Abstract 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.

BackgroundIt 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.MethodsImmortalized 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.ResultsObtained 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.ConclusionsThese 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.

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-β1, 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.

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.

Abstract 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.

The microbiota is a complex ecosystem of active microorganisms resident in the body of mammals. Although the majority of these microorganisms resides in the distal gastrointestinal tract, high-throughput DNA sequencing technology has made possible to understand that several other tissues of the human body host their own microbiota, even those once considered sterile, such as lung tissue. These bacterial communities have important functions in maintaining a healthy body state, preserving symbiosis with the host immune system, which generates protective responses against pathogens and regulatory pathways that sustain the tolerance to commensal microbes. Toll-like receptors (TLRs) are critical in sensing the microbiota, maintaining the tolerance or triggering an immune response through the direct recognition of ligands derived from commensal microbiota or pathogenic microbes. Lately, it has been highlighted that the resident microbiota influences the initiation and development of cancer and its response to therapies and that specific changes in the number and distribution of taxa correlate with the existence of cancers in various tissues. However, the knowledge of functional activity and the meaning of microbiome changes remain limited. This review summarizes the current findings on the function of TLRs as sensors of the microbiota and highlighted their modulation as a reflection of tumor-associated changes in commensal microbiota. The data available to date suggest that commensal “onco-microbes” might be able to break the tolerance of TLRs and become complicit in cancer by sustaining its growth.

Background1,25-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.MethodsCytotoxicity 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.ResultsIn 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.ConclusionsTogether, 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.

E-cadherin, an epithelial-to-mesenchymal transition (EMT) marker, is coupled to actin cytoskeleton and distributes cell forces acting on cells. Since YAP transduces mechanical signals involving actin cytoskeleton, we aimed to investigate the relationship between YAP and mechanical cues in pancreatic ductal adenocarcinoma (PDAC) cell lines, characterized by different EMT-related phenotypes, cultured in 2D monolayers and 3D spheroids. We observed that the YAP/p-YAP ratio was reduced in HPAC and MIA PaCa-2 cell lines and remained unchanged in BxPC-3 cells when cultured in a 3D setting. CTGF and CYR61 gene expression were down-regulated in all PDAC 3D compared to 2D cultures, without any significant effect following actin cytoskeleton inhibition by Cytochalasin B (CyB) treatment. Moreover, LATS1 mRNA, indicating the activation of the Hippo pathway, was not influenced by CyB and differed in all PDAC cell lines having different EMT-related phenotype but a similar pattern of CTGF and CYR61 expression. Although the role of YAP modulation in response to mechanical cues in cancer cells remains to be completely elucidated, our results suggest that cell arrangement and phenotype can determine variable outcomes to mechanical stimuli in PDAC cells. Moreover, it is possible to speculate that YAP and Hippo pathways may act as parallel and not exclusive inputs that, converging at some points, may impact cell behavior.

Summary: 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. : Le Noci et al. reveal that modulation of pulmonary microbiota by antibiotic or probiotic aerosolization decreases tumor growth in the lung. Antibiotic treatment induces a reduction of immunosuppressive cells in the lung, while probiotic administration promotes maturation of resident antigen-presenting cells. Keywords: lung microbiota, cancer immunosurveillance, aerosolization, antibiotics, probiotics, mouse models, immunosuppression and/or suppressor cells

Mechanotransduction is the ability of cells to translate mechanical stimuli into biochemical signals that can ultimately influence gene expression, cell morphology and cell fate. Tenocytes are responsible for tendon mechanical adaptation converting mechanical stimuli imposed during mechanical loading, thus affecting extracellular matrix homeostasis. Since we previously demonstrated that MD-Tissue, an injectable collagen-based medical compound containing swine-derived collagen as the main component, is able to affect tenocyte properties, the aim of this study was to analyze whether the effects triggered by MD-Tissue were based on mechanotransduction-related mechanisms. For this purpose, MD-Tissue was used to coat Petri dishes and cytochalasin B was used to deprive tenocytes of mechanical stimulation mediated by the actin cytoskeleton. Cell morphology, migration, collagen turnover pathways and the expression of key mechanosensors were analyzed by morphological and molecular methods. Our findings confirm that MD-Tissue affects collagen turnover pathways and favors cell migration and show that the MD-Tissue-induced effect represents a mechanical input involving the mechanotransduction machinery. Overall, MD-Tissue, acting as a mechanical scaffold, could represent an effective medical device for a novel therapeutic, regenerative and rehabilitative approach to favor tendon healing in tendinopathies.

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.

Epithelial-to-mesenchymal transition (EMT) is a step-wise process observed in normal and tumor cells leading to a switch from epithelial to mesenchymal phenotype. In tumors, EMT provides cancer cells with a metastatic phenotype characterized by E-cadherin down-regulation, cytoskeleton reorganization, motile and invasive potential. E-cadherin down-regulation is known as a key event during EMT. However, E-cadherin expression can be influenced by the different experimental settings and environmental stimuli so that the paradigm of EMT based on the loss of E-cadherin determining tumor cell behavior and fate often becomes an open question. In this review, we aimed at focusing on some critical points in order to improve the knowledge of the dynamic role of epithelial cells plasticity in EMT and, specifically, address the role of E-cadherin as a marker for the EMT axis.

Three-dimensional (3D) cell cultures allow the mimic of functions of living tissues and provide key information encoded in tissue architecture. Considered the pivotal role of epithelial-to-mesenchymal transition (EMT) in carcinoma progression, including prostate cancer (PCa), we aimed at investigating the effect of the 3D arrangement on the expression of some key markers of EMT in cultured human prostate cancer (PCa) cells, to better understand PCa cell behavior. PC3 and DU145 PCa cells were cultured in RPMI cell culture medium either in 2D-monolayers or in 3D-spheroids. The main EMT markers E-cadherin, N-cadherin, α-smooth muscle actin (αSMA), vimentin, Snail, Slug, Twist and Zeb1 were evaluated by confocal microscopy, real-time PCR and Western blot. Confocal microscopy revealed that E-cadherin was similarly expressed at the cell boundaries on the plasma membrane of PCa cells grown in 2D-monolayers, as well as in 3D-spheroids, but resulted up-regulated in 3D-spheroids, compared to 2D-monolayers, at the mRNA and protein level. Moreover, markers of the mesenchymal phenotype were expressed at very low levels in 3D-spheroids, suggesting important differences in the phenotype of PCa cells grown in 3D-spheroids or in 2D-monolayers. Considered as a whole, our findings contribute to a clarification of the role of EMT in PCa and confirm that a 3D cell culture model could provide deeper insight into the understanding of the biology of PCa.

The extracellular matrix (ECM) in the tumor microenvironment modulates the cancer cell phenotype, especially in pancreatic ductal adenocarcinoma (PDAC), a tumor characterized by an intense desmoplastic reaction. Because the epithelial-to-mesenchymal transition (EMT), a process that provides cancer cells with a metastatic phenotype, plays an important role in PDAC progression, the authors aimed to explore in vitro the interactions between human PDAC cells and ECM components of the PDAC microenvironment, focusing on the expression of EMT markers and matrix metalloproteinases (MMPs) that are able to digest the basement membrane during tumor invasion. EMT markers and the invasive potential of HPAF-II, HPAC, and PL45 cells grown on different ECM substrates (fibronectin, laminin, and collagen) were analyzed. While N-cadherin, αSMA, and type I collagen were not significantly affected by ECM components, the E-cadherin/β-catenin complex was highly expressed in all the experimental conditions, and E-cadherin was upregulated by collagen in PL45 cells. Cell migration was unaffected by fibronectin and delayed by laminin. In contrast, collagen significantly stimulated cell migration and the secretion of MMPs. This study’s results showed that ECM components impacted cell migration and invasive potential differently. Collagen exerted a more evident effect, providing new insights into the understanding of the intricate interplay between ECM molecules and cancer cells, in order to find novel therapeutic targets for PDAC treatment.

We recently reported that peritumoral CpG-ODN treatment, activating TLR-9 expressing cells in tumor microenvironment, induces modulation of genes involved in DNA repair and sensitizes cancer cells to DNA-damaging cisplatin treatment. Here, we investigated whether this treatment induces modulation of miRNAs in tumor cells and their relevance to chemotherapy response. Array analysis identified 20 differentially expressed miRNAs in human IGROV-1 ovarian tumor cells from CpG-ODN-treated mice versus controls (16 down- and 4 up-regulated). Evaluation of the role of the 3 most differentially expressed miRNAs on sensitivity to cisplatin of IGROV-1 cells revealed significantly increased cisplatin cytotoxicity upon ectopic expression of hsa-miR-302b (up-modulated in our array), but no increased effect upon reduced expression of hsa-miR-424 or hsa-miR-340 (down-modulated in our array). Accordingly, hsa-miR-302b expression was significantly associated with time to relapse or overall survival in two data sets of platinum-treated ovarian cancer patients. Use of bio-informatics tools identified 19 mRNAs potentially targeted by hsa-miR-302b, including HDAC4 gene, which has been reported to mediate cisplatin sensitivity in ovarian cancer. Both HDAC4 mRNA and protein levels were significantly reduced in IGROV-1 cells overexpressing hsa-miR-302b. Altogether, these findings indicate that hsa-miR-302b acts as a "chemosensitizer" in human ovarian carcinoma cells and may represent a biomarker able to predict response to cisplatin treatment. Moreover, the identification of miRNAs that improve sensitivity to chemotherapy provides the experimental underpinning for their possible future clinical use.