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.

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.

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.

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.

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.

Melanoma Stem Cells Educate Neutrophils to Support Cancer Progression.

BACKGROUND: It is now well-established that cancer stem cells (CSCs) can support melanoma progression by reshaping the tumor immune microenvironment. However, the molecular mechanisms underlying the crosstalk between melanoma SCs and cancer-associated neutrophils have not been elucidated yet. METHODS: The aim of the present study was to unravel the role of melanoma SCs in neutrophil polarization. HL60 neutrophil-like (dHL60) cells were treated with conditioned medium from A375 melanoma SCs (CSC-CM), and their phenotype was investigated. RESULTS: We demonstrated that CSC-CM could specifically activate immune cells by increasing CD66b and CD11b expression. In particular, we revealed that A375 CSCs could release various soluble factors, namely TGF-ß, IL-6, and IL-8, able to promote the recruitment of neutrophils and their switch toward an N2 phenotype characterized by the activation of ERK, STAT3, and P38 pathways and the overexpression of CXCR2 and NF-kB. Moreover, after exposure to CSC-CM, dHL60 cells exhibited enhanced ROS production and NET release, without undergoing cell death; increased secretion of MMP-9 and pro-inflammatory cytokines was also observed. Finally, CSC-CM-activated neutrophils endowed A375 cells with stemness traits, stimulating both sphere formation and ABCG2 expression. CONCLUSION: Collectively, our results suggest that melanoma SCs can prime neutrophils to support cancer progression.

Mechanical Cues, E-Cadherin Expression and Cell "Sociality" Are Crucial Crossroads in Determining Pancreatic Ductal Adenocarcinoma Cells Behavior.

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.

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.

Toll Like Receptors as Sensors of the Tumor Microbial Dysbiosis: Implications in Cancer Progression.

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.

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.

In Vitro 3D Cultures to Model the Tumor Microenvironment.

It is now well established that the tumor microenvironment plays a key role in determining cancer growth, metastasis and drug resistance. Thus, it is fundamental to understand how cancer cells interact and communicate with their stroma and how this crosstalk regulates disease initiation and progression. In this setting, 3D cell cultures have gained a lot of interest in the last two decades, due to their ability to better recapitulate the complexity of tumor microenvironment and therefore to bridge the gap between 2D monolayers and animal models. Herein, we present an overview of the 3D systems commonly used for studying tumor-stroma interactions, with a focus on recent advances in cancer modeling and drug discovery and testing.

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.

Effect of acetylsalicylic acid on inflamed adipose tissue. Insulin resistance and hepatic steatosis in a mouse model of diet-induced obesity.

AIMS: Obesity represents a global health problem. Excessive caloric intake promotes the release of inflammatory mediators by hypertrophic adipocytes and obesity-induced inflammation is now recognized as a risk factor for the development of several diseases, such as cardiovascular diseases, insulin resistance, type-II diabetes, liver steatosis and cancer. Since obesity causes inflammation, we tested the ability of acetylsalicylic acid (ASA), a potent anti-inflammatory drug, in counteracting this inflammatory process and in mitigating obesity-associated health complications. MAIN METHODS: Mice were fed with standard (SD) or high fat diet (HFD) for 3 months and then treated with acetylsalicylic acid for the subsequent two months. We then analyzed the metabolic and inflammatory status of their adipose and liver tissue by histological, molecular and biochemical analysis. KEY FINDINGS: Although ASA did not exert any effect on body weight, quantification of adipocyte size revealed that the drug slightly reduced adipocyte hypertrophy, however not sufficient so as to induce weight loss. Most importantly, ASA was able to improve insulin resistance. Gene expression profiles of pro- and anti-inflammatory cytokines as well as the expression of macrophage and lymphocyte markers revealed that HFD led to a marked macrophage accumulation in the adipose tissue and an increase of several pro-inflammatory cytokines, a situation almost completely reverted after ASA administration. In addition, liver steatosis caused by HFD was completely abrogated by ASA treatment. SIGNIFICANCE: ASA can efficiently ameliorate pathological conditions usually associated with obesity by inhibiting the inflammatory process occurring in the adipose tissue.

The Collagen-Based Medical Device MD-Tissue Acts as a Mechanical Scaffold Influencing Morpho-Functional Properties of Cultured Human Tenocytes.

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.

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.