Mouse dendritic cells in the steady state: Hypoxia, autophagy, and stem cell factor.

Dendritic cells (DCs) are innate immune cells with a central role in immunity and tolerance. Under steady-state, DCs are scattered in tissues as resting cells. Upon infection or injury, DCs get activated and acquire the full capacity to prime antigen-specific CD4+ and CD8+ T cells, thus bridging innate and adaptive immunity. By secreting different sets of cytokines and chemokines, DCs orchestrate diverse types of immune responses, from a classical proinflammatory to an alternative pro-repair one. DCs are highly heterogeneous, and physiological differences in tissue microenvironments greatly contribute to variations in DC phenotype. Oxygen tension is normally low in some lymphoid areas, including bone marrow (BM) hematopoietic niches; nevertheless, the possible impact of tissue hypoxia on DC physiology has been poorly investigated. We assessed whether DCs are hypoxic in BM and spleen, by staining for hypoxia-inducible-factor-1α subunit (HIF-1α), the master regulator of hypoxia-induced response, and pimonidazole (PIM), a hypoxic marker, and by flow cytometric analysis. Indeed, we observed that mouse DCs have a hypoxic phenotype in spleen and BM, and showed some remarkable differences between DC subsets. Notably, DCs expressing membrane c-kit, the receptor for stem cell factor (SCF), had a higher PIM median fluorescence intensity (MFI) than c-kit- DCs, both in the spleen and in the BM. To determine whether SCF (a.k.a. kit ligand) has a role in DC hypoxia, we evaluated molecular pathways activated by SCF in c-kit+ BM-derived DCs cultured in hypoxic conditions. Gene expression microarrays and gene set enrichment analysis supported the hypothesis that SCF had an impact on hypoxia response and inhibited autophagy-related gene sets. Our results suggest that hypoxic response and autophagy, and their modulation by SCF, can play a role in DC homeostasis at the steady state, in agreement with our previous findings on SCF's role in DC survival.

Durable CD8 T Cell Memory against SARS-CoV-2 by Prime/Boost and Multi-Dose Vaccination: Considerations on Inter-Dose Time Intervals.

Facing the COVID-19 pandemic, anti-SARS-CoV-2 vaccines were developed at unprecedented pace, productively exploiting contemporary fundamental research and prior art. Large-scale use of anti-SARS-CoV-2 vaccines has greatly limited severe morbidity and mortality. Protection has been correlated with high serum titres of neutralizing antibodies capable of blocking the interaction between the viral surface protein spike and the host SARS-CoV-2 receptor, ACE-2. Yet, vaccine-induced protection subsides over time, and breakthrough infections are commonly observed, mostly reflecting the decay of neutralizing antibodies and the emergence of variant viruses with mutant spike proteins. Memory CD8 T cells are a potent weapon against viruses, as they are against tumour cells. Anti-SARS-CoV-2 memory CD8 T cells are induced by either natural infection or vaccination and can be potentially exploited against spike-mutated viruses. We offer here an overview of current research about the induction of anti-SARS-CoV-2 memory CD8 T cells by vaccination, in the context of prior knowledge on vaccines and on fundamental mechanisms of immunological memory. We focus particularly on how vaccination by two doses (prime/boost) or more (boosters) promotes differentiation of memory CD8 T cells, and on how the time-length of inter-dose intervals may influence the magnitude and persistence of CD8 T cell memory.

Biological Effects of Cyclin-Dependent Kinase Inhibitors Ribociclib, Palbociclib and Abemaciclib on Breast Cancer Bone Microenvironment.

The CDK4/6 inhibitors (CDKi) palbociclib, ribociclib, and abemaciclib are currently approved in combination with anti-estrogen therapy for the treatment of advanced and/or metastatic hormone receptor-positive/HER2-neu-negative breast cancer patients. Given the high incidence of bone metastases in this population, we investigated and compared the potential effects of palbociclib, ribociclib, and abemaciclib on the breast cancer bone microenvironment. Primary osteoclasts (OCs) and osteoblasts (OBs) were obtained from human monocyte and mesenchymal stem cells, respectively. OC function was evaluated by tartrate-resistant acid phosphatase assay and real-time PCR; OB activity was assessed by an alizarin red assay. OB/breast cancer co-culture models were generated via the seeding of MCF-7 cells on a layer of OBs, and tumor cell proliferation was analyzed using flow cytometry. Here, we showed that ribociclib, palbociclib, and abemaciclib exerted similar inhibitory effects on the OC differentiation and expression of bone resorption markers without affecting OC viability. On the other hand, the three CDKi did not affect the ability of OB to produce bone matrix, even if the higher doses of palbociclib and abemaciclib reduced the OB viability. In OB/MCF-7 co-culture models, palbociclib demonstrated a lower anti-tumor effect than ribociclib and abemaciclib. Overall, our results revealed the direct effects of CDKi on the tumor bone microenvironment, highlighting differences potentially relevant for clinical practice.

OMIP-079: Cell cycle of CD4+ and CD8+ naïve/memory T cell subsets, and of Treg cells from mouse spleen.

A multicolor flow cytometry panel was designed and optimized to define the following nine mouse T cell subsets: Treg (CD3+ CD4+ CD8- FoxP3+ ), CD4+ T naïve (CD3+ CD4+ CD8- FoxP3- CD44int/low CD62L+ ), CD4+ T central memory (CD3+ CD4+ CD8- FoxP3- CD44high CD62L+ ), CD4+ T effector memory (CD3+ CD4+ CD8- FoxP3- CD44high CD62L- ), CD4+ T EMRA (CD3+ CD4+ CD8- FoxP3- CD44int/low CD62L- ), CD8+ T naïve (CD3+ CD8+ CD4- CD44int/low CD62L+ ), CD8+ T central memory (CD3+ CD8+ CD4- CD44high CD62L+ ), CD8+ T effector memory (CD3+ CD8+ CD4- CD44high CD62L- ), and CD8+ T EMRA (CD3+ CD8+ CD4- CD44int/low CD62L- ). In each T cell subset, a dual staining for Ki-67 expression and DNA content was employed to distinguish the following cell cycle phases: G0 (Ki67- , with 2n DNA), G1 (Ki67+ , with 2n DNA), and S-G2 /M (Ki67+ , with 2n < DNA ≤ 4n). This panel was established for the analysis of mouse (C57BL/6J) spleen.

Dendritic cells modulate c-kit expression on the edge between activation and death.

Dendritic cells (DCs) are key players in immunity and tolerance. Some DCs express c-kit, the receptor for stem cell factor (SCF), nevertheless c-kit functional role and the regulation of its expression in DCs are incompletely defined. We recently demonstrated that autocrine SCF sustains a pro-survival circuit, and that SCF increases phospho-AKT in c-kit+ mouse bone marrow-derived DCs (BMdDCs). Herein we observed that CpG and PolyI:C, two stimuli mimicking bacterial and viral nucleic acids respectively, strongly inhibited c-kit expression by BMdDCs and spleen DCs in vitro and in vivo. Experiments in IFNARI-/- mice showed that IFN-I pathway was required for c-kit down-regulation in cDC1s, but only partially supported it in cDC2s. Furthermore, CpG and PolyI:C strongly inhibited c-kit mRNA expression. In agreement with the reduced c-kit levels, SCF pro-survival activity was impaired. Thus in the presence of exogenously provided SCF, either PolyI:C or CpG induced spleen DC death in 2 days, while at earlier times IL-6 and IL-12 production were slightly increased. In contrast, SCF improved survival of unstimulated spleen DCs expressing high c-kit levels. Our studies suggest that c-kit down-modulation is a previously neglected component of DC response to CpG and PolyI:C, regulating DC survival and ultimately tuning immune response.

Antigen-specific CD8 T cells in cell cycle circulate in the blood after vaccination.

Although clonal expansion is a hallmark of adaptive immunity, the location(s) where antigen-responding T cells enter cell cycle and complete it have been poorly explored. This lack of knowledge stems partially from the limited experimental approaches available. By using Ki67 plus DNA staining and a novel strategy for flow cytometry analysis, we distinguished antigen-specific CD8 T cells in G0 , in G1 and in S-G2 /M phases of cell cycle after intramuscular vaccination of BALB/c mice with antigen-expressing viral vectors. Antigen-specific cells in S-G2 /M were present at early times after vaccination in lymph nodes (LNs), spleen and, surprisingly, also in the blood, which is an unexpected site for cycling of normal non-leukaemic cells. Most proliferating cells had high scatter profile and were undetected by current criteria of analysis, which under-estimated up to 6 times antigen-specific cell frequency in LNs. Our discovery of cycling antigen-specific CD8 T cells in the blood opens promising translational perspectives.

Circulating receptor activator of nuclear factor kappa-B ligand (RANKL) levels predict response to immune checkpoint inhibitors in advanced non-small cell lung cancer (NSCLC).

BACKGROUND: Receptor activator of nuclear factor kappa-B ligand (RANKL) can directly promote tumor growth and indirectly support tumor immune evasion by altering the tumor microenvironment and immune cell responses. This study aimed to assess the prognostic significance of soluble RANKL in patients with advanced non-small cell lung cancer (NSCLC) receiving programmed cell death 1 (PD1)/programmed death-ligand 1 (PDL1) checkpoint inhibitor therapy. METHODS: Plasma RANKL levels were measured in 100 patients with advanced NSCLC without bone metastases undergoing monotherapy with PD1/PDL1 checkpoint inhibitors. To establish the optimal cut-off value, we used the Cutoff Finder package in R. Survival curves for four distinct patient groups, according to their RANKL and PDL1 levels (high or low), were generated using the Kaplan-Meier method and compared with the log-rank test. The Cox regression model calculated HRs and 95% CIs for overall survival (OS) and progression-free survival (PFS). RESULTS: The optimal RANKL cut-off was established at 280.4 pg/mL, categorizing patients into groups with high or low RANKL levels. A significant association was observed between increased RANKL concentrations and decreased survival rates at 24 months, only within the subgroup expressing high levels of PDL1 (p=0.002). Additionally, low RANKL levels in conjunction with elevated PDL1 expression correlated with improved PFS (median 22 months, 95% CI 6.70 to 50 vs median 4 months, 95% CI 3.0 to 7.30, p=0.009) and OS (median 26 months, 95% CI 20 to not reached vs median 7 months, 95% CI 6 to 13, p=0.003), indicating RANKL's potential as an indicator of adverse prognosis in these patients. Multivariate analysis identified RANKL as an independent negative prognostic factor for both PFS and OS, regardless of other clinicopathological features. CONCLUSION: These results highlight the prognostic and predictive value of RANKL specifically in patients with high PDL1 expression.

Improved memory CD8 T cell response to delayed vaccine boost is associated with a distinct molecular signature.

Effective secondary response to antigen is a hallmark of immunological memory. However, the extent of memory CD8 T cell response to secondary boost varies at different times after a primary response. Considering the central role of memory CD8 T cells in long-lived protection against viral infections and tumors, a better understanding of the molecular mechanisms underlying the changing responsiveness of these cells to antigenic challenge would be beneficial. We examined here primed CD8 T cell response to boost in a BALB/c mouse model of intramuscular vaccination by priming with HIV-1 gag-encoding Chimpanzee adenovector, and boosting with HIV-1 gag-encoding Modified Vaccinia virus Ankara. We found that boost was more effective at day(d)100 than at d30 post-prime, as evaluated at d45 post-boost by multi-lymphoid organ assessment of gag-specific CD8 T cell frequency, CD62L-expression (as a guide to memory status) and in vivo killing. RNA-sequencing of splenic gag-primed CD8 T cells at d100 revealed a quiescent, but highly responsive signature, that trended toward a central memory (CD62L+) phenotype. Interestingly, gag-specific CD8 T cell frequency selectively diminished in the blood at d100, relative to the spleen, lymph nodes and bone marrow. These results open the possibility to modify prime/boost intervals to achieve an improved memory CD8 T cell secondary response.

Corrigendum: Improved memory CD8 T cell response to delayed vaccine boost is associated with a distinct molecular signature.

[This corrects the article DOI: 10.3389/fimmu.2023.1043631.].

Network approach in liquidomics landscape.

Tissue-based biopsy is the present main tool to explore the molecular landscape of cancer, but it also has many limits to be frequently executed, being too invasive with the risk of side effects. These limits and the ability of cancer to constantly evolve its genomic profile, have recently led to the need of a less invasive and more accurate alternative, such as liquid biopsy. By searching Circulating Tumor Cells and residues of their nucleic acids or other tumor products in body fluids, especially in blood, but also in urine, stools and saliva, liquid biopsy is becoming the future of clinical oncology. Despite the current lack of a standardization for its workflows, that makes it hard to be reproduced, liquid biopsy has already obtained promising results for cancer screening, diagnosis, prognosis, and risk of recurrence.Through a more accessible molecular profiling of tumors, it could become easier to identify biomarkers predictive of response to treatment, such as EGFR mutations in non-small cell lung cancer and KRAS mutations in colorectal cancer, or Microsatellite Instability and Mismatch Repair as predictive markers of pembrolizumab response.By monitoring circulating tumor DNA in longitudinal repeated sampling of blood we could also predict Minimal Residual Disease and the risk of recurrence in already radically resected patients.In this review we will discuss about the current knowledge of limitations and strengths of the different forms of liquid biopsies for its inclusion in normal cancer management, with a brief nod to their newest biomarkers and its future implications.

Extensive plasma proteomic profiling revealed receptor activator of nuclear factor kappa-Β ligand (RANKL) as emerging biomarker of nivolumab clinical benefit in patients with metastatic renal cell carcinoma.

BACKGROUND: The advent of immune checkpoint inhibitors (ICIs) have led to a paradigm change in the management of metastatic renal cell carcinoma (mRCC), nevertheless, the benefit of treatment is confined to a limited proportion of patients. Therefore, the identification of predictive biomarkers for response to ICIs represents an unmet clinical need. Here, we performed a large-scale plasma proteomic profile of patients with mRCC, treated with nivolumab, to identify soluble molecules potentially associated with clinical benefit. METHODS: We analyzed the levels of 507 soluble molecules in the pretreatment plasma of 16 patients with mRCC (discovery set) who received nivolumab therapy as a single agent. The ELISA assay was performed to confirm the protein level of candidate biomarkers associated to clinical benefit in 15 patients with mRCC (validation set). Survival curves of complete cohort were estimated by the Kaplan-Meier method and compared with the log-rank test. RESULTS: Out of 507 screened molecules, 135 factors were selected as expressed above background and 12 of them were significantly overexpressed in patients who did not benefit from treatment (non-responders (NR)) compared with responders (R) group. After multiplicity adjustment, receptor activator of nuclear factor kappa-Β ligand (RANKL) was the only molecule that retained the statistical significance (false discovery rate: 0.023). RANKL overexpression in NR patients was confirmed both in discovery (median NR: 528 pg/mL vs median R: 288 pg/mL, p=0.011) and validation set (median NR: 440 pg/mL vs median R: 253 pg/mL, p<0.001). Considering the complete cohort of patients (discovery+validation set), significantly higher RANKL levels were found in patients who primarily progressed from treatment compared with those who had a partial response (p=0.003) or stable disease (p=0.006). Moreover, patients with low RANKL levels had significant improvements in progression-free survival (median 14.0 months vs 3.4 months, p=0.004) and overall survival (median not reached vs 30.1 months, p=0.003). CONCLUSIONS: Our exploratory study suggests RANKL as a novel independent biomarker of response and survival in patients with mRCC treated with nivolumab.

Osteoblast Secretome Modulated by Abiraterone Treatment Affects Castration Resistant Prostate Cancer Cell Proliferation.

Abiraterone is a selective inhibitor of androgen biosynthesis approved for the treatment of metastatic patients affected by castration-resistant or castration-sensitive prostate cancer. Intriguingly, clinical data revealed that abiraterone also delayed disease progression in bone improving bone-related endpoints. Our group has previously demonstrated in vitro a direct effect of abiraterone on osteoclast and osteoblast function suggesting its ability to modulate bone microenvironment. Here, we performed an extensive proteomic analysis to investigate how abiraterone influences osteoblast cell secretome and, consequently, osteoblast/prostate cancer cells interaction. A panel of 507 soluble molecules were analyzed in osteoblast conditioned media (OCM) obtained from osteoblast treated or not with abiraterone. Subsequently, OCM was added to prostate cancer cells to investigate its potential effect on prostate cancer cell proliferation and androgen receptor (AR) activation status. Out of 507 screened molecules, 39 of them were differentially expressed in OCM from osteoblasts treated with abiraterone (OCM ABI) compared to OCM obtained from untreated OBs (OCM CTRL). Pathway enrichment analysis revealed that abiraterone down-modulated the release of specific osteoblast soluble factors, positively associated with cell proliferation pathways (false discovery rate adjusted p-value = 0.0019). In vitro validation data showed that OCM ABI treatment significantly reduced cancer proliferation in C4-2B cells (p = 0.022), but not in AR- negative PC-3 cells. Moreover, we also found a reduction in AR activation in C4-2B cells (p = 0.017) confirming the "indirect" anti-tumor AR-dependent effect of abiraterone mediated by osteoblasts. This study provides the first evidence of an additional antitumor effect of abiraterone through the modulation of multiple osteoblast proliferative signals.

Changes in bone turnover markers in patients without bone metastases receiving immune checkpoint inhibitors: An exploratory analysis.

Immune checkpoint inhibitors (ICIs) has revolutionized the treatment of different advanced solid tumors, but most patients develop severe immune-related adverse events (irAEs). Although a bi-directional crosstalk between bone and immune systems is widely described, the effect of ICIs on the skeleton is poorly investigated. Here, we analyze the changes in plasma levels of type I collagen C-terminal telopeptide (CTX-I) and N-terminal propeptide of type I procollagen (PINP), reference makers of bone turnover, in patients treated with ICIs and their association with clinical outcome. A series of 44 patients affected by advanced non-small cell lung cancer or renal cell carcinoma, without bone metastases, and treated with ICIs as monotherapy were enrolled. CTX-I and PINP plasma levels were assessed at baseline and after 3 months of ICIs treatment by ELISA kits. A significant increase of CTX-I with a concomitant decreasing trend towards the reduction of PINP was observed after 3 months of treatment. Intriguingly, CTX-I increase was associated with poor prognosis in terms of treatment response and survival. These data suggest a direct relationship between ICIs treatment, increased osteoclast activity and potential fracture risk. Overall, this study reveals that ICIs may act as triggers for skeletal events, and if confirmed in larger prospective studies, it would identify a new class of skeletal-related irAEs.

Large-Scale Profiling of Extracellular Vesicles Identified miR-625-5p as a Novel Biomarker of Immunotherapy Response in Advanced Non-Small-Cell Lung Cancer Patients.

Immune checkpoint inhibitors (ICIs) are largely used in the treatment of patients with advanced non-small-cell lung cancer (NSCLC). Novel biomarkers that provide biological information that could be useful for clinical management are needed. In this respect, extracellular vesicles (EV)-associated microRNAs (miRNAs) that are the principal vehicle of intercellular communication may be important sources of biomarkers. We analyzed the levels of 799 EV-miRNAs in the pretreatment plasma of 88 advanced NSCLC patients who received anti-PD-1 therapy as single agent. After data normalization, we used a two-step approach to identify candidate biomarkers associated to both objective response (OR) by RECIST and longer overall survival (OS). Univariate and multivariate analyses including known clinicopathologic variables and new findings were performed. In our cohort, 24/88 (27.3%) patients showed OR by RECIST. Median OS in the whole cohort was 11.5 months. In total, 196 EV-miRNAs out 799 were selected as expressed above background. After multiplicity adjustment, abundance of EV-miR-625-5p was found to be correlated with PD-L1 expression and significantly associated to OR by RECIST (p = 0.0366) and OS (p = 0.0031). In multivariate analysis, PD-L1 staining and EV-miR-625-5p levels were constantly associated to OR and OS. Finally, we showed that EV-miR-625-5p levels could discriminate patients with longer survival, in particular in the class expressing PD-L1 ≥50%. EV-miRNAs represent a source of relevant biomarkers. EV-miR-625-5p is an independent biomarker of response and survival in ICI-treated NSCLC patients, in particular in patients with PD-L1 expression ≥50%.

Osteoblasts Promote Prostate Cancer Cell Proliferation Through Androgen Receptor Independent Mechanisms.

Patients with metastatic prostate cancer frequently develop bone metastases that elicit significant skeletal morbidity and increased mortality. The high tropism of prostate cancer cells for bone and their tendency to induce the osteoblastic-like phenotype are a result of a complex interplay between tumor cells and osteoblasts. Although the role of osteoblasts in supporting prostate cancer cell proliferation has been reported by previous studies, their precise contribution in tumor growth remains to be fully elucidated. Here, we tried to dissect the molecular signaling underlining the interactions between castration-resistant prostate cancer (CRPC) cells and osteoblasts using in vitro co-culture models. Transcriptomic analysis showed that osteoblast-conditioned media (OCM) induced the overexpression of genes related to cell cycle in the CRPC cell line C4-2B but, surprisingly, reduced androgen receptor (AR) transcript levels. In-depth analysis of AR expression in C4-2B cells after OCM treatment showed an AR reduction at the mRNA (p = 0.0047), protein (p = 0.0247), and functional level (p = 0.0029) and, concomitantly, an increase of C4-2B cells in S-G2-M cell cycle phases (p = 0.0185). An extensive proteomic analysis revealed in OCM the presence of some molecules that reduced AR activation, and among these, Matrix metalloproteinase-1 (MMP-1) was the only one able to block AR function (0.1 ng/ml p = 0.006; 1 ng/ml p = 0.002; 10 ng/ml p = 0.0001) and, at the same time, enhance CRPC proliferation (1 ng/ml p = 0.009; 10 ng/ml p = 0.033). Although the increase of C4-2B cell growth induced by MMP-1 did not reach the proliferation levels observed after OCM treatment, the addition of Vorapaxar, an MMP-1 receptor inhibitor (Protease-activated receptor-1, PAR-1), significantly reduced C4-2B cell cycle (0.1 µM p = 0.014; 1 µM p = 0.0087). Overall, our results provide a novel AR-independent mechanism of CRPC proliferation and suggest that MMP-1/PAR-1 could be one of the potential pathways involved in this process.

Antitumor Effect of Cabozantinib in Bone Metastatic Models of Renal Cell Carcinoma.

BACKGROUND: The presence of bone metastases in renal cell carcinoma (RCC) negatively affects patients' survival. Data from clinical trials has highlighted a significant benefit of cabozantinib in bone metastatic RCC patients. Here, we evaluated the antitumor effect of cabozantinib in coculture models of renal cell carcinoma (RCC) and osteoblasts (OBs) to investigate whether and how its antiproliferative activity is influenced by OBs. METHODS: Bone/RCC models were generated, coculturing green fluorescent protein (GFP)-tagged Caki-1 and 786-O cells with human primary OBs in a "cell-cell contact" system. RCC proliferation and the OB molecular profile were evaluated after the cabozantinib treatment. RESULTS: The Caki-1 cell proliferation increased in the presence of OBs (p < 0.0001), while the 786-O cell growth did not change in the coculture with the OBs. The cabozantinib treatment reduced the proliferation of both the Caki-1 (p < 0.0001) and 786-O (p = 0.03) cells cocultured with OBs. Intriguingly, the inhibitory potency of cabozantinib was higher when Caki-1 cells grew in presence of OBs compared to a monoculture (p < 0.001), and this was similar in 786-O cells alone or cocultured with OBs. Moreover, the OB pretreatment with cabozantinib "indirectly" inhibited Caki-1 cell proliferation (p = 0.040) without affecting 786-O cell growth. Finally, we found that cabozantinib was able to modulate the OB gene and molecular profile inhibiting specific proliferative signals that, in turn, could affect RCC cell growth. CONCLUSIONS: Overall, the "direct" effect of cabozantinib on OBs "indirectly" increased its antitumor activity in metastatic RCC Caki-1 cells but not in the primary 786-O model.

A DNA/Ki67-Based Flow Cytometry Assay for Cell Cycle Analysis of Antigen-Specific CD8 T Cells in Vaccinated Mice.

The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations. Bromodeoxyuridine (BrdU) marks cells that are in or recently completed S-phase, and carboxyfluorescein succinimidyl ester (CFSE) detects daughter cells after division. However, these dyes do not allow identification of the cell cycle phase at the time of analysis. An alternative approach is to exploit Ki67, a marker that is highly expressed by cells in all phases of the cell cycle except the quiescent phase G0. Unfortunately, Ki67 does not allow further differentiation as it does not separate cells in S-phase that are committed to mitosis from those in G1 that can remain in this phase, proceed into cycling, or move into G0. Here, we describe a flow cytometric method for capturing a "snapshot" of T cells in different cell cycle phases in mouse secondary lymphoid organs. The method combines Ki67 and DNA staining with major histocompatibility complex (MHC)-peptide-multimer staining and an innovative gating strategy, allowing us to successfully differentiate between antigen-specific CD8 T cells in G0, in G1 and in S-G2/M phases of the cell cycle in the spleen and draining lymph nodes of mice after vaccination with viral vectors carrying the model antigen gag of human immunodeficiency virus (HIV)-1. Critical steps of the method were the choice of the DNA dye and the gating strategy to increase the assay sensitivity and to include highly activated/proliferating antigen-specific T cells that would have been missed by current criteria of analysis. The DNA dye, Hoechst 33342, enabled us to obtain a high-quality discrimination of the G0/G1 and G2/M DNA peaks, while preserving membrane and intracellular staining. The method has great potential to increase knowledge about T cell response in vivo and to improve immuno-monitoring analysis.

Integrin alpha5 in human breast cancer is a mediator of bone metastasis and a therapeutic target for the treatment of osteolytic lesions.

Bone metastasis remains a major cause of mortality and morbidity in breast cancer. Therefore, there is an urgent need to better select high-risk patients in order to adapt patient's treatment and prevent bone recurrence. Here, we found that integrin alpha5 (ITGA5) was highly expressed in bone metastases, compared to lung, liver, or brain metastases. High ITGA5 expression in primary tumors correlated with the presence of disseminated tumor cells in bone marrow aspirates from early stage breast cancer patients (n = 268; p = 0.039). ITGA5 was also predictive of poor bone metastasis-free survival in two separate clinical data sets (n = 855, HR = 1.36, p = 0.018 and n = 427, HR = 1.62, p = 0.024). This prognostic value remained significant in multivariate analysis (p = 0.028). Experimentally, ITGA5 silencing impaired tumor cell adhesion to fibronectin, migration, and survival. ITGA5 silencing also reduced tumor cell colonization of the bone marrow and formation of osteolytic lesions in vivo. Conversely, ITGA5 overexpression promoted bone metastasis. Pharmacological inhibition of ITGA5 with humanized monoclonal antibody M200 (volociximab) recapitulated inhibitory effects of ITGA5 silencing on tumor cell functions in vitro and tumor cell colonization of the bone marrow in vivo. M200 also markedly reduced tumor outgrowth in experimental models of bone metastasis or tumorigenesis, and blunted cancer-associated bone destruction. ITGA5 was not only expressed by tumor cells but also osteoclasts. In this respect, M200 decreased human osteoclast-mediated bone resorption in vitro. Overall, this study identifies ITGA5 as a mediator of breast-to-bone metastasis and raises the possibility that volociximab/M200 could be repurposed for the treatment of ITGA5-positive breast cancer patients with bone metastases.

Current and Emerging Biomarkers Predicting Bone Metastasis Development.

Bone is one of the preferential sites of distant metastases from malignant tumors, with the highest prevalence observed in breast and prostate cancers. Patients with bone metastases (BMs) may experience skeletal-related events, such as severe bone pain, pathological fractures, spinal cord compression, and hypercalcemia, with negative effects on the quality of life. In the last decades, a deeper understanding of the molecular mechanisms underlying the BM onset has been gained, leading to the development of bone-targeting agents. So far, most of the research has been focused on the pathophysiology and treatment of BM, with only relatively few studies investigating potential predictors of risk for BM development. The ability to select such "high-risk" patients could allow early identification of those most likely to benefit from interventions to prevent or delay BM. This review summarizes several evidences for the potential use of specific biomarkers able to predict early the BM development.

Correction to: Liquid biopsy and tumor heterogeneity in metastatic solid tumors: the potentiality of blood samples.

An amendment to this paper has been published and can be accessed via the original article.