Pro-tumor Activity Research Articles

Potential involvement of IL-32 in cell-to-cell communication between macrophages and hepatoblastoma.

This study investigated the expression of interleukin 32 (IL-32) in hepatoblastoma, the most common primary pediatric liver tumor, and its possible roles in tumorigenesis. IL-32 expression was investigated in two hepatoblastoma cell lines (Hep G2 and HuH 6) in the steady state and after co-culture with macrophages by RNA-seq analysis and RT-qPCR, and after stimulation with chemotherapy. Cultured macrophages were stimulated by IL-32 isoforms followed by RT-qPCR and western blot analysis. IL-32 immunohistochemical staining (IHC) was performed using specimens from 21 hepatoblastoma patients. Clustering analysis was also performed using scRNA-seq data downloaded from Gene Expression Omnibus. The IL-32 gene is expressed by hepatoblastoma cell lines; expression is upregulated by paracrine cell-cell communication with macrophages, also by carboplatin and etoposide. IL-32 causes protumor activation of macrophages with upregulation of PD-L1, IDO-1, IL-6, and IL-10. In the patient pool, IHC was positive only in 48% of cases. However, in the downloaded dataset, IL-32 gene expression was negative. IL-32 was detected in hepatoblastoma cell lines, but not in all hepatoblastoma patients. We hypothesized that stimulation such as chemotherapy might induce expression of IL-32, which might be a critical mediator of chemoresistance in hepatoblastoma through inducing protumor activation in macrophages.

Immune checkpoint blockade therapy mitigates systemic inflammation and affects cellular FLIP-expressing monocytic myeloid-derived suppressor cells in non-progressor non-small cell lung cancer patients

ABSTRACT Cancer cells favor the generation of myeloid cells with immunosuppressive and inflammatory features, including myeloid-derived suppressor cells (MDSCs), which support tumor progression. The anti-apoptotic molecule, cellular FLICE (FADD-like interleukin-1β-converting enzyme)-inhibitory protein (c-FLIP), which acts as an important modulator of caspase-8, is required for the development and function of monocytic (M)-MDSCs. Here, we assessed the effect of immune checkpoint inhibitor (ICI) therapy on systemic immunological landscape, including FLIP-expressing MDSCs, in non-small cell lung cancer (NSCLC) patients. Longitudinal changes in peripheral immunological parameters were correlated with patients’ outcome. In detail, 34 NSCLC patients were enrolled and classified as progressors (P) or non-progressors (NP), according to the RECIST evaluation. We demonstrated a reduction in pro-inflammatory cytokines such as IL-8, IL-6, and IL-1β in only NP patients after ICI treatment. Moreover, using t-distributed stochastic neighbor embedding (t-SNE) and cluster analysis, we characterized in NP patients a significant increase in the amount of lymphocytes and a slight contraction of myeloid cells such as neutrophils and monocytes. Despite this moderate ICI-associated alteration in myeloid cells, we identified a distinctive reduction of c-FLIP expression in M-MDSCs from NP patients concurrently with the first clinical evaluation (T1), even though NP and P patients showed the same level of expression at baseline (T0). In agreement with the c-FLIP expression, monocytes isolated from both P and NP patients displayed similar immunosuppressive functions at T0; however, this pro-tumor activity was negatively influenced at T1 in the NP patient cohort exclusively. Hence, ICI therapy can mitigate systemic inflammation and impair MDSC-dependent immunosuppression.

Mechanistic Insights into the Roles of the IL-17/IL-17R Families in Pancreatic Cancer.

The members of the cytokine interleukin 17 (IL-17) family, along with their receptors (IL-17R), are vital players in a range of inflammatory diseases and cancer. Although generally regarded as proinflammatory, the effects they exhibit on cancer progression are a double-edged sword, with both antitumor and protumor activities being discovered. There is growing evidence that the IL-17 signaling pathways have significant impacts on the tumor microenvironment (TME), immune response, and inflammation in various types of cancer, including pancreatic cancer. However, the detailed mechanistic functions of the IL-17/IL-17R families in pancreatic cancer were rarely systematically elucidated. This review considers the role of the IL-17/IL-17R families in inflammation and tumor immunity and elaborates on the mechanistic functions and correlations of these members with pathogenesis, progression, and chemoresistance in pancreatic cancer. By summarizing the advanced findings on the role of IL-17/IL17R family members and IL-17 signaling pathways at the molecular level, cellular level, and disease level in pancreatic cancer, this review provides an in-depth discussion on the potential of IL-17/IL-17R as prognostic markers and therapeutic targets in pancreatic cancer.

Pegylated Liposomal Alendronate Biodistribution, Immune Modulation, and Tumor Growth Inhibition in a Murine Melanoma Model.

While tumor-associated macrophages (TAM) have pro-tumoral activity, the ablation of macrophages in cancer may be undesirable since they also have anti-tumoral functions, including T cell priming and activation against tumor antigens. Alendronate is a potent amino-bisphosphonate that modulates the function of macrophages in vitro, with potential as an immunotherapy if its low systemic bioavailability can be addressed. We repurposed alendronate in a non-leaky and long-circulating liposomal carrier similar to that of the clinically approved pegylated liposomal doxorubicin to facilitate rapid clinical translation. Here, we tested liposomal alendronate (PLA) as an immunotherapeutic agent for cancer in comparison with a standard of care immunotherapy, a PD-1 immune checkpoint inhibitor. We showed that the PLA induced bone marrow-derived murine non-activated macrophages and M2-macrophages to polarize towards an M1-functionality, as evidenced by gene expression, cytokine secretion, and lipidomic profiles. Free alendronate had negligible effects, indicating that liposome encapsulation is necessary for the modulation of macrophage activity. In vivo, the PLA showed significant accumulation in tumor and tumor-draining lymph nodes, sites of tumor immunosuppression that are targets of immunotherapy. The PLA remodeled the tumor microenvironment towards a less immunosuppressive milieu, as indicated by a decrease in TAM and helper T cells, and inhibited the growth of established tumors in the B16-OVA melanoma model. The improved bioavailability and the beneficial effects of PLA on macrophages suggest its potential application as immunotherapy that could synergize with T-cell-targeted therapies and chemotherapies to induce immunogenic cell death. PLA warrants further clinical development, and these clinical trials should incorporate tumor and blood biomarkers or immunophenotyping studies to verify the anti-immunosuppressive effect of PLA in humans.

Suspected oncologic adverse reactions associated with interleukin-23 inhibitors in EudraVigilance: Comparative study and gender distribution.

Psoriasis is a chronic inflammatory skin disease characterized by plaque formation. Interleukin (IL)-23 is upregulated in psoriatic lesions and is thought to be a major regulator of the Th17 pathway in psoriasis pathogenesis. Three monoclonal antibodies targeting the IL-23p19 subunit, guselkumab, tildrakizumab, and risankizumab, have been approved for psoriasis therapy. The balance between cytokines IL-23 and IL-12 can affect antitumor and pro-tumor immune activities, and patients with psoriasis may have higher rates of cancer than the general population. Moreover, a chronic inflammatory state typical of psoriasis may induce protumorigenic effects, however, the potential risk of malignancy in patients taking these drugs remains largely unknown. This study investigated the occurrence of malignancies as suspected adverse reactions (SARs) potentially associated with IL-23 inhibitors by analyzing real-world data from the European EudraVigilance database. Although indicatory, these real-world data seem to confirm the potential association between the IL-23 inhibitors risankizumab and tildrakizumab, and the occurrence of SARs linked to cancer in patients with psoriasis and, according to a gender perspective, they show that this relationship is asymmetrically distributed between women and men, with a clear prevalence of oncologic SARs in men.

The Relationship Between Lymphocyte Subtypes with Clinicopathological Features and Prognosis of Prostate Cancer in Patients Undergoing Radical Prostatectomy.

The immune system has an essential role in the development of cancer by showing both anti-tumor and pro-tumor activities. Understanding the immune func- tion of patients with malignancy is of clinical importance for the evaluation, treatment, and prognosis of the disease. We aimed to evaluate lymphocyte subtypes in peripheral blood samples of prostate cancer patients and their relationship with clinicopathologi- cal features and prognosis. One hundred thirty-seven patients who underwent open radical prosta- tectomy were included in our study. The percentages of CD3+T lymphocyte, CD19+ B lymphocyte, CD16/56 natural killer cells, CD4+ helper T lymphocyte, CD8+ cytotoxic T lymphocyte, and CD45 total lymphocyte were evaluated for each patient using the blood sample taken into a hemogram tube before surgery. The pathological stage was T2 for 64 of the cases and T3 for 73. The mean follow-up period of the patients was 12.81 ± 6.20 months. The CD3+/CD4+ counts of the patients with pathological stage T2 were found to be statistically significantly higher than stage T3. There was a statistically significant negative correlation between the prostate-specific antigen levels and CD3+/CD4+ percentages of the patients. There was no statistical significance between the percentages of lymphocyte subtypes and the presence of surgical margin, biochemical recurrence, adjuvant therapy, and cancer upgrade. We consider that the increase in the pathological stage and prostate-spe- cific antigen value and the decrease in the number of CD4+ T lymphocyte subtypes may be prognostic markers in prostate cancer patients.

Long noncoding RNA HOXC-AS3 remodels lipid metabolism and promotes the proliferation of transformed macrophages in the glioma stem cell microenvironment by regulating the hnRNPA1/CaM axis

Metabolism remodelling of macrophages in the glioblastoma microenvironment contributes to immunotherapeutic resistance. However, glioma stem cell (GSC)-initiated lipid metabolism remodelling of transformed macrophages (tMΦs) and its effect on the glioblastoma microenvironment have not been fully elucidated. Total cholesterol (TC) levels and lipid metabolism enzyme expression in macrophages in the GSC microenvironment were evaluated and found that the TC levels of tMΦs were increased, and the expression of the lipid metabolism enzymes calmodulin (CaM), apolipoprotein E (ApoE), and liver X receptor (LXR) was upregulated. Knockdown of HOXC-AS3 led to a decrease in the proliferation, colony formation, invasiveness, and tumorigenicity of tMΦs. Downregulation of CaM resulted in a decline in TC levels. HOXC-AS3 overexpression led to increases in both CaM expression levels and TC levels in tMΦs. RNA pull down and mass spectrometry experiments were conducted and heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) was screened as the HOXC-AS3 binding proteins related to lipid metabolism. RIP and RNA pull down assays verified that HOXC-AS3 can form a complex with hnRNPA1. Knockdown of hnRNPA1 downregulated CaM expression; however, downregulation of HOXC-AS3 did not affect hnRNPA1 expression.TMΦs underwent lipid metabolism remodelling induced by GSC via the HOXC-AS3/hnRNPA1/CaM pathway, which enhanced the protumor activities of tMΦs, and may serve as a potential metabolic intervening target to improve glioblastoma immunotherapy.

IRE1a-Induced FilaminA Phosphorylation Enhances Migration of Mesenchymal Stem Cells Derived from Multiple Myeloma Patients.

Multiple myeloma (MM) is an aggressive malignancy that shapes, during its progression, a pro-tumor microenvironment characterized by altered protein secretion and the gene expression of mesenchymal stem cells (MSCs). In turn, MSCs from MM patients can exert an high pro-tumor activity and play a strong immunosuppressive role. Here, we show, for the first time, greater cell mobility paralleled by the activation of FilaminA (FLNA) in MM-derived MSCs, when compared to healthy donor (HD)-derived MSCs. Moreover, we suggest the possible involvement of the IRE1a-FLNA axis in the control of the MSC migration process. In this way, IRE1a can be considered as a good target candidate for MM therapy, considering its pro-survival, pro-osteoclast and chemoresistance role in the MM microenvironment. Our results suggest that IRE1a downregulation could also interfere with the response of MSCs to MM stimuli, possibly preventing cell-cell adhesion-mediated drug resistance. In addition, further investigations harnessing IRE1a-FLNA interaction could improve the homing efficiency of MSC as cell product for advanced therapy applications.

Pro-Tumor Activity of Endogenous Nitric Oxide in Anti-Tumor Photodynamic Therapy: Recently Recognized Bystander Effects.

Various studies have revealed that several cancer cell types can upregulate inducible nitric oxide synthase (iNOS) and iNOS-derived nitric oxide (NO) after moderate photodynamic treatment (PDT) sensitized by 5-aminolevulinic acid (ALA)-induced protoporphyrin-IX. As will be discussed, the NO signaled cell resistance to photokilling as well as greater growth and migratory aggressiveness of surviving cells. On this basis, it was predicted that diffusible NO from PDT-targeted cells in a tumor might enhance the growth, migration, and invasiveness of non- or poorly PDT-targeted bystander cells. This was tested using a novel approach in which ALA-PDT-targeted cancer cells on a culture dish were initially segregated from non-targeted bystander cells of the same type via impermeable silicone-rimmed rings. Several hours after LED irradiation, the rings were removed, and both cell populations were analyzed in the dark for various responses. After a moderate extent of targeted cell killing (~25%), bystander proliferation and migration were evaluated, and both were found to be significantly enhanced. Enhancement correlated with iNOS/NO upregulation in surviving PDT-targeted cancer cells in the following cell type order: PC3 > MDA-MB-231 > U87 > BLM. If occurring in an actual PDT-challenged tumor, such bystander effects might compromise treatment efficacy by stimulating tumor growth and/or metastatic dissemination. Mitigation of these and other negative NO effects using pharmacologic adjuvants that either inhibit iNOS transcription or enzymatic activity will be discussed.

Antibody-based soluble and membrane-bound TWEAK mimicking agonists with FcγR-independent activity.

Fibroblast growth factor (FGF)-inducible 14 (Fn14) activates the classical and alternative NFκB (nuclear factor 'kappa-light-chain-enhancer' of activated B-cells) signaling pathway but also enhances tumor necrosis factor (TNF)-induced cell death. Fn14 expression is upregulated in non-hematopoietic cells during tissue injury and is also often highly expressed in solid cancers. In view of the latter, there were and are considerable preclinical efforts to target Fn14 for tumor therapy, either by exploiting Fn14 as a target for antibodies with cytotoxic activity (e.g. antibody-dependent cellular cytotoxicity (ADCC)-inducing IgG variants, antibody drug conjugates) or by blocking antibodies with the aim to interfere with protumoral Fn14 activities. Noteworthy, there are yet no attempts to target Fn14 with agonistic Fc effector function silenced antibodies to unleash the proinflammatory and cell death-enhancing activities of this receptor for tumor therapy. This is certainly not at least due to the fact that anti-Fn14 antibodies only act as effective agonists when they are presented bound to Fcγ receptors (FcγR). Thus, there are so far no antibodies that robustly and selectively engage Fn14 signaling without triggering unwanted FcγR-mediated activities. In this study, we investigated a panel of variants of the anti-Fn14 antibody 18D1 of different valencies and domain architectures with respect to their inherent FcγR-independent ability to trigger Fn14-associated signaling pathways. In contrast to conventional 18D1, the majority of 18D1 antibody variants with four or more Fn14 binding sites displayed a strong ability to trigger the alternative NFκB pathway and to enhance TNF-induced cell death and therefore resemble in their activity soluble (TNF)-like weak inducer of apoptosis (TWEAK), one form of the natural occurring ligand of Fn14. Noteworthy, activation of the classical NFκB pathway, which naturally is predominately triggered by membrane-bound TWEAK but not soluble TWEAK, was preferentially observed with a subset of constructs containing Fn14 binding sites at opposing sites of the IgG scaffold, e.g. IgG1-scFv fusion proteins. A superior ability of IgG1-scFv fusion proteins to trigger classical NFκB signaling was also observed with the anti-Fn14 antibody PDL192 suggesting that we identified generic structures for Fn14 antibody variants mimicking soluble and membrane-bound TWEAK.

Tumor-Associated Macrophages in Multiple Myeloma: Key Role in Disease Biology and Potential Therapeutic Implications.

Multiple myeloma (MM) is characterized by multiple relapse and, despite the introduction of novel therapies, the disease becomes ultimately drug-resistant. The tumor microenvironment (TME) within the bone marrow niche includes dendritic cells, T-cytotoxic, T-helper, reactive B-lymphoid cells and macrophages, with a complex cross-talk between these cells and the MM tumor cells. Tumor-associated macrophages (TAM) have an important role in the MM pathogenesis, since they could promote plasma cells proliferation and angiogenesis, further supporting MM immune evasion and progression. TAM are polarized towards M1 (classically activated, antitumor activity) and M2 (alternatively activated, pro-tumor activity) subtypes. Many studies demonstrated a correlation between TAM, disease progression, drug-resistance and reduced survival in lymphoproliferative neoplasms, including MM. MM plasma cells in vitro could favor an M2 TAM polarization. Moreover, a possible correlation between the pro-tumor effect of M2 TAM and a reduced sensitivity to proteasome inhibitors and immunomodulatory drugs was hypothesized. Several clinical studies confirmed CD68/CD163 double-positive M2 TAM were associated with increased microvessel density, chemoresistance and reduced survival, independently of the MM stage. This review provided an overview of the biology and clinical relevance of TAM in MM, as well as a comprehensive evaluation of a potential TAM-targeted immunotherapy.

Prognostic value of macrophage vasculiform mimicry in the evaluation of uveal melanoma progression

Introduction Uveal melanoma (UM) is a malignant neoplasm of the vascular tract of the eye, prone to hematogenous metastasis, regardless of the type of treatment performed. Prediction and probability of UM metastasis is one of the main tasks of onco-ophthalmology. We decribed an alternative method of tumor blood supply due to the process of vasculo-like mimicry (VM) with the formation of pseudovascular structures of tumor cells is described. At the same time, the participation of the microenvironment in the processes of VM and metastasis is currently debatable and relevant.Purpose of the work was to evaluate the prognostic role of macrophage vasculiform mimicry in the progression of chorioid melanoma based on the histological and immunohistochemical characteristics of surgical material.Materials and methods The surgical material of choroidal melanoma after enucleation for the period 2013-2018 was studied. Histological, histochemical (PAS reaction) and immunohistochemical studies were carried out using markers CD68 (macrophages), CD 34, CD31, D2-40 for the purpose of qualitative and quantitative assessment of macrophages in the stroma of tumors, large vessels and pseudovessels in the tumor.Results The ability of macrophages to form vasculo-like spaces in choroidal melanoma has been discovered and described. In growth with macrometastases, more phage representatives are recorded than in tissue samples of non-metastasizing melanoma. the volume of the macroscopic mass of the ulcer and the growth rate of phages were registered as the inverse average in terms of the strength of the correlation. In group studies with metastases, an increase in the volume of pseudovascular volumes and the number of phages is recorded.Discussion The macrophage microenvironment in a tumor can play a dual role: antitumor and protumor. The ability of macrophages for VM in UM has been described. The results of our macrostudy confirm the protumor activity of phages and their important role in tumor metastasis.Conclusion In-depth study of the ways of pseudovascular space formation not only by the cells of the tumor itself, but also by its microenvironment, helps to open new directions in the study of predictors of UM metastasis and outline approaches to the development of new directions of anti-tumortherapy.

Combination therapy with anti-programmed cell death 1 antibody plus angiokinase inhibitor exerts synergistic antitumor effect against malignant mesothelioma via tumor microenvironment modulation

Malignant pleural mesothelioma (MPM) is an asbestos-related fatal malignant neoplasm. Although there has been no reliable chemotherapeutic regimen other than combination therapy of cisplatin and pemetrexed for two decades, combination of ipilimumab plus nivolumab brought about a better outcome in patients with MPM. Thus, cancer immunotherapy using immune checkpoint inhibitor (ICI) is expected to play a central role in the treatment of MPM. To maximize the antitumor effect of ICI, we evaluated whether nintedanib, an antiangiogenic agent, could augment the antitumor effect of anti-programmed cell death 1 (PD-1) antibody (Ab). Although nintedanib could not inhibit the proliferation of mesothelioma cells in vitro, it significantly suppressed the growth of mesothelioma allografts in mice. Moreover, combination therapy with anti-PD-1 Ab plus nintedanib reduced tumor burden more dramatically compared with nintedanib monotherapy via inducing remarkable necrosis in MPM allografts. Nintedanib did not promote the infiltration of CD8+ T cells within the tumor when used alone or in combination with anti-PD-1 Ab but it independently decreased the infiltration of tumor-associated macrophages (TAMs). Moreover, immunohistochemical analysis and ex vivo study using bone marrow-derived macrophages (BMDMs) showed that nintedanib could polarize TAMs from M2 to M1 phenotype. These results indicated that nintedanib had a potential to suppress protumor activity of TAMs both numerically and functionally. On the other hand, ex vivo study revealed that nintedanib upregulated the expression of PD-1 and PD-ligand 1 (PD-L1) in BMDMs and mesothelioma cells, respectively, and exhibited the impairment of phagocytic activity of BMDMs against mesothelioma cells. Co-administration of anti-PD-1 Ab may reactivate phagocytic activity of BMDMs by disrupting nintedanib-induced immunosuppressive signal via binding between PD-1 on BMDMs and PD-L1 on mesothelioma cells. Collectively, combination therapy of anti-PD-1 Ab plus nintedanib enhances the antitumor activity compared with respective monotherapy and can become a novel therapeutic option for patients with MPM.

A pathogenicity locus of Streptococcus gallolyticus subspecies gallolyticus

Streptococcus gallolyticus subspecies gallolyticus (Sgg) is known to be strongly associated with colorectal cancer (CRC). Recent functional studies further demonstrated that Sgg actively stimulates CRC cell proliferation and promotes the development of colon tumors. However, the Sgg factors important for the pro-proliferative and pro-tumor activities of Sgg remain unclear. Here, we identified a chromosomal locus in Sgg strain TX20005. Deletion of this locus significantly reduced Sgg adherence to CRC cells and abrogated the ability of Sgg to stimulate CRC cell proliferation. Thus, we designate this locus as the Sgg pathogenicity-associated region (SPAR). More importantly, we found that SPAR is important for Sgg pathogenicity in vivo. In a gut colonization model, mice exposed to the SPAR deletion mutant showed significantly reduced Sgg load in the colonic tissues and fecal materials, suggesting that SPAR contributes to the colonization capacity of Sgg. In a mouse model of CRC, deletion of SPAR abolished the ability of Sgg to promote the development of colon tumors growth. Taken together, these results highlight SPAR as a critical pathogenicity determinant of Sgg.

Active immunization with a structurally aggregated PD-L1 antigen breaks T and B immune tolerance in non-human primates and exhibits in vivo anti-tumoral effects in immunocompetent mouse tumor models

Despite the clinical success of the programmed death ligand 1 (PD-L1) blocking therapy in cancer treatment, only a subset of patients exhibits durable responses, therefore further exploration of other immunotherapeutic alternatives are needed. This paper reported the development of the PKPD-L1Vac vaccine, a new protein vaccine candidate that uses aluminum phosphate as an adjuvant and as an antigen the extracellular domain of human PD-L1 fused to a 47 amino-terminal portion of the LpdA protein from N. meningitides (PKPD-L1). The PKPD-L1 antigen has different physical and biological characteristics than those found in the natural molecule and in others PD-L1 vaccine candidates. The quimeric protein has a reduced binding capacity to the PD-1 and CD80 receptors to decrease their pro-tumoral activity. Besides, the distinctive feature of the PKPD-L1 polypeptide to be structurally aggregated could be desirable for its immunogenic properties. PKPD-L1Vac elicited anti-PD-L1-specific IgG antibodies and T lymphocyte-mediated immunity in mice and non-human primates. The vaccine administration demonstrated antitumor activity on CT-26 and B16–F10 primary tumor models in mice. Moreover, the immunization with PKPD-L1Vac increased the tumor-infiltrating lymphocytes and decreased the proportion of CD3+CD8+PD1+high anergic T cells in CT-26 tumor tissues, suggesting that the vaccine may remodel the tumor microenvironment. In summary, the PKPD-L1Vac vaccine exhibits very promising preclinical results and deserves to move forward to a phase I clinical trial.

Extracellular Lactic Acidosis of the Tumor Microenvironment Drives Adipocyte-to-Myofibroblast Transition Fueling the Generation of Cancer-Associated Fibroblasts.

Lactic acidosis characterizes the tumor microenvironment (TME) and is involved in the mechanisms leading to cancer progression and dissemination through the reprogramming of tumor and local host cells (e.g., endothelial cells, fibroblasts, and immune cells). Adipose tissue also represents a crucial component of the TME which is receiving increasing attention due to its pro-tumoral activity, however, to date, it is not known whether it could be affected by the acidic TME. Now, emerging evidence from chronic inflammatory and fibrotic diseases underlines that adipocytes may give rise to pathogenic myofibroblast-like cells through the adipocyte-to-myofibroblast transition (AMT). Thus, our study aimed to investigate whether extracellular acidosis could affect the AMT process, sustaining the acquisition by adipocytes of a cancer-associated fibroblast (CAF)-like phenotype with a pro-tumoral activity. To this purpose, human subcutaneous adipose-derived stem cells committed to adipocytes (acADSCs) were cultured under basal (pH 7.4) or lactic acidic (pH 6.7, 10 mM lactate) conditions, and AMT was evaluated with quantitative PCR, immunoblotting, and immunofluorescence analyses. We observed that lactic acidosis significantly impaired the expression of adipocytic markers while inducing myofibroblastic, pro-fibrotic, and pro-inflammatory phenotypes in acADSCs, which are characteristic of AMT reprogramming. Interestingly, the conditioned medium of lactic acidosis-exposed acADSC cultures was able to induce myofibroblastic activation in normal fibroblasts and sustain the proliferation, migration, invasion, and therapy resistance of breast cancer cells in vitro. This study reveals a previously unrecognized relationship between lactic acidosis and the generation of a new CAF-like cell subpopulation from adipocytic precursor cells sustaining tumor malignancy.

An exosome-related lncRNA signature correlates with prognosis, immune microenvironment, and therapeutic responses in hepatocellular carcinoma

BackgroundExosomes act as essential modulators of cancer development and progression in hepatocellular carcinoma. However, little is known about the potential prognostic value and underlying molecular features of exosome-related long non-coding RNAs. MethodsGenes associated with exosome biogenesis, exosome secretion, and exosome biomarkers were collected. Exosome-related lncRNA modules were identified using PCA and WGCNA analysis. A prognostic model based on data from the TCGA, GEO, NODE, and ArrayExpress was developed and validated. A comprehensive analysis of the genomic landscape, functional annotation, immune profile, and therapeutic responses underlying the prognostic signature was performed on multi-omics data, and bioinformatics methods were also applied to predict potential drugs for patients with high risk scores. qRT-PCR was used to validate the differentially expressed lncRNAs in normal and cancer cell lines. ResultsTwenty-six hub lncRNAs were identified as highly correlated with exosomes and overall survival and were used for prognosis modeling. Three cohorts consistently showed higher scores in the high-risk group, with an AUC greater than 0.7 over time. These higher scores implied poorer overall survival, higher genomic instability, higher tumor purity, higher tumor stemness, pro-tumor pathway activation, lower anti-tumor immune cell and tertiary lymphoid structure infiltration, and poor responses to immune checkpoint blockade therapy and transarterial chemoembolization therapy. ConclusionThrough developing an exosome-related lncRNA predictor for HCC patients, we revealed the clinical relevance of exosome-related lncRNAs and their potential as prognostic biomarkers and therapeutic response predictors.

Proteome integral solubility alteration high-throughput proteomics assay identifies Collectin-12 as a non-apoptotic microglial caspase-3 substrate

Caspases are a family of proteins mostly known for their role in the activation of the apoptotic pathway leading to cell death. In the last decade, caspases have been found to fulfill other tasks regulating the cell phenotype independently to cell death. Microglia are the immune cells of the brain responsible for the maintenance of physiological brain functions but can also be involved in disease progression when overactivated. We have previously described non-apoptotic roles of caspase-3 (CASP3) in the regulation of the inflammatory phenotype of microglial cells or pro-tumoral activation in the context of brain tumors. CASP3 can regulate protein functions by cleavage of their target and therefore could have multiple substrates. So far, identification of CASP3 substrates has been performed mostly in apoptotic conditions where CASP3 activity is highly upregulated and these approaches do not have the capacity to uncover CASP3 substrates at the physiological level. In our study, we aim at discovering novel substrates of CASP3 involved in the normal regulation of the cell. We used an unconventional approach by chemically reducing the basal level CASP3-like activity (by DEVD-fmk treatment) coupled to a Mass Spectrometry screen (PISA) to identify proteins with different soluble amounts, and consequently, non-cleaved proteins in microglia cells. PISA assay identified several proteins with significant change in their solubility after DEVD-fmk treatment, including a few already known CASP3 substrates which validated our approach. Among them, we focused on the Collectin-12 (COLEC12 or CL-P1) transmembrane receptor and uncovered a potential role for CASP3 cleavage of COLEC12 in the regulation of the phagocytic capacity of microglial cells. Taken together, these findings suggest a new way to uncover non-apoptotic substrates of CASP3 important for the modulation of microglia cell physiology.

Heterogeneity of Cholangiocarcinoma Immune Biology

Cholangiocarcinomas (CCAs) are aggressive tumors arising along the biliary tract epithelium, whose incidence and mortality are increasing. CCAs are highly desmoplastic cancers characterized by a dense tumor microenvironment (TME), in which each single component plays a fundamental role in shaping CCA initiation, progression and resistance to therapies. The crosstalk between cancer cells and TME can affect the recruitment, infiltration and differentiation of immune cells. According to the stage of the disease and to intra- and inter-patient heterogeneity, TME may contribute to either protumoral or antitumoral activities. Therefore, a better understanding of the effect of each immune cell subtype may open the path to new personalized immune therapeutic strategies for the management of CCA. In this review, we describe the role of immune cells in CCA initiation and progression, and their crosstalk with both cancer-associated fibroblasts (CAFs) and the cancer-stem-cell-like (CSC) niche.

Evaluating tumor cell- and T cell-derived extracellular vesicles as potential biomarkers of cancer and immune cell competence

ABSTRACT Introduction Extracellular vesicles (EVs) produced by tumors, also called tumor-derived exosomes (TEX), have been implicated in inducing immune cell suppression in vitro and in vivo. The development of a novel category of noninvasive biomarkers for precision oncology remains an unmet need, and TEX emerge as a promising liquid tumor biopsy component. Areas covered TEX play a critical role in monitoring cancer presence/progression and in reprograming of anti-tumor effector T cells to producers of EVs with pro-tumor activity. TEX are a subset of circulating EVs. Their separation by immune capture from EVs derived from nonmalignant cells allows for TEX phenotypic/functional assessments. TEX cross-talking with CD3(+) T cells induce the release of CD3(+) small EV (sEV), whose cargo of suppressor proteins resembles that of TEX and further contributes to cancer-induced immune suppression. While TEX recapitulate the genetic/molecular phenotype of tumor cells, CD3(+) sEV might serve as ‘T cell liquid biopsy.’ Expert opinion Preclinical explorations of the role in cancer body fluids of TEX and CD3(+) sEV as cancer biomarkers suggest that these EV subsets may qualify as liquid tumor biopsy noninvasive components in the near future. Their potential to simultaneously serve as noninvasive liquid tumor biopsy and T cell biopsy remains to be validated in future clinical trials.