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Many cancer patients do not benefit from PD-L1/PD-1 blockade immunotherapies. PD-1 and LAG-3 co-upregulation in T-cells is one of the major mechanisms of resistance by establishing a highly dysfunctional state in T-cells. To identify shared features associated to PD-1/LAG-3 dysfunctionality in human cancers and T-cells, multiomic expression profiles were obtained for all TCGA cancers immune infiltrates. A PD-1/LAG-3 dysfunctional signature was found which regulated immune, metabolic, genetic, and epigenetic pathways, but especially a reinforced negative regulation of the TCR signalosome. These results were validated in T-cell lines with constitutively active PD-1, LAG-3 pathways and their combination. A differential analysis of the proteome of PD-1/LAG-3 T-cells showed a specific enrichment in ubiquitin ligases participating in E3 ubiquitination pathways. PD-1/LAG-3 co-blockade inhibited CBL-B expression, while the use of a bispecific drug in clinical development also repressed C-CBL expression, which reverted T-cell dysfunctionality in lung cancer patients resistant to PD-L1/PD-1 blockade. The combination of CBL-B-specific small molecule inhibitors with anti-PD-1/anti-LAG-3 immunotherapies demonstrated notable therapeutic efficacy in models of lung cancer refractory to immunotherapies, overcoming PD-1/LAG-3 mediated resistance., (© 2024. The Author(s).)

CX3CL1 is one of the 50 up-to-date identified and characterized chemokines. While other chemokines are produced as small, secreted proteins, CX3CL1 (fractalkine) is synthetized as a transmembrane protein which also leads to a soluble form produced as a result of proteolytic cleavage. The membrane-bound protein and the soluble forms exhibit different biological functions. While the role of the fractalkine/CX3CR1 signaling axis was described in the nervous system and was also related to the migration of leukocytes to sites of inflammation, its actions are controversial in cancer progression and anti-tumor immunity. In the present review, we first describe the known biology of fractalkine concerning its action through its cognate receptor, but also its role in the activation of different integrins. The second part of this review is dedicated to its role in cancer where we discuss its role in anti-cancer or procarcinogenic activities.

Background: Previous studies have shown that functional systemic immunity is required for the efficacy of PD-1/PD-L1 blockade immunotherapies in cancer. Hence, systemic reprogramming of immunosuppressive dysfunctional myeloid cells could overcome resistance to cancer immunotherapy., Methods: Reprogramming of tumour-associated myeloid cells with oleuropein was studied by quantitative differential proteomics, phenotypic and functional assays in mice and lung cancer patients. Combinations of oleuropein and two different delivery methods of anti-PD-1 antibodies were tested in colorectal cancer tumour models and in immunotherapy-resistant lung cancer models., Results: Oleuropein treatment reprogrammed monocytic and granulocytic myeloid-derived suppressor cells, and tumour-associated macrophages towards differentiation of immunostimulatory subsets. Oleuropein regulated major differentiation programmes associated to immune modulation in myeloid cells, which potentiated T cell responses and PD-1 blockade. PD-1 antibodies were delivered by two different strategies, either systemically or expressed within tumours using a self-amplifying RNA vector. Combination anti-PD-1 therapies with oleuropein increased tumour infiltration by immunostimulatory dendritic cells in draining lymph nodes, leading to systemic antitumour T cell responses. Potent therapeutic activities were achieved in colon cancer and lung cancer models resistant to immunotherapies, even leading to complete tumour regression., Discussion: Oleuropein significantly improves the outcome of PD-1/PD-L1 blockade immunotherapy strategies by reprogramming myeloid cells., (© 2024. The Author(s).)

Myeloid-derived suppressor cells (MDSCs) are major promoters of progression and metastasis in cancer. MDSCs inhibit the anti-tumor immune response through multiple mechanisms. The main MDSC functions in cancer are related to the inactivation of T cells and the establishment of an immunosuppressive tumor microenvironment (TME) through the production of pro-inflammatory cytokines, among other mechanisms. MDSCs are phenotypically similar to conventional myeloid cells, so their identification is challenging. Moreover, they infiltrate the tumors in limited numbers, and their purification from within the tumors is technically difficult and makes their study a challenge. Therefore, several ex vivo differentiation methods have been established. Our differentiation method leads to MDSCs that closely model tumor-infiltrating counterparts. In this protocol, MDSCs are differentiated from bone marrow precursors by incubation in differentiation medium produced by murine tumor cell lines engineered to constitutively express granulocyte-monocyte colony stimulating factor (GM-CSF). These ex vivo-generated MDSC subsets show high fidelity compared to their natural tumor-infiltrated counterparts. Moreover, the high yields of purification from these ex vivo differentiated MDSC enable their use for validation of new treatments in high-throughput assays. In this chapter we describe the engineering of a stable cell line overexpressing GM-CSF, followed by production and collection of conditioned media supporting MDSC differentiation. Finally, we detail the isolation procedure of bone marrow cells and the specific MDSC differentiation protocol., Competing Interests: Disclosures The authors declare no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Background: Identification of predictive biomarkers to Immune checkpoint inhibitors (ICIs) in head and neck cancer (HNSCC) is an unmet need., Methods: This was a prospective observational study including 25 patients with HNSCC treated with immunotherapy or chemotherapy after a prior platinum-based regimen. Low density neutrophils (LDNs) and serum markers were analyzed., Results: In the immunotherapy cohort, patients with high LDN levels had a shorter progression free survival (PFS) (1.8 months vs. 10.9 months; *p = 0.034). Also, progressors showed higher percentage of LDNs compared to non-progressors although significance was not reached (mean 20.68% vs. 4.095%, p = 0.0875). These findings were not replicated in patients treated with chemotherapy. High levels of interleukin-7 (IL7) were correlated with a significantly longer overall survival (OS) (13.47 months 3.51 vs. months, *p = 0.013)., Conclusions: High baseline circulating LDNs and low IL7 could identify a subset of patients intrinsically refractory to ICIs as monotherapy in HNSCC., (© 2023 Wiley Periodicals LLC.)

Resistance of melanoma to targeted therapy and immunotherapy is linked to metabolic rewiring. Here, we show that increased fatty acid oxidation (FAO) during prolonged BRAF inhibitor (BRAFi) treatment contributes to acquired therapy resistance in mice. Targeting FAO using the US Food and Drug Administration-approved and European Medicines Agency-approved anti-anginal drug ranolazine (RANO) delays tumour recurrence with acquired BRAFi resistance. Single-cell RNA-sequencing analysis reveals that RANO diminishes the abundance of the therapy-resistant NGFR hi neural crest stem cell subpopulation. Moreover, by rewiring the methionine salvage pathway, RANO enhances melanoma immunogenicity through increased antigen presentation and interferon signalling. Combination of RANO with anti-PD-L1 antibodies strongly improves survival by increasing antitumour immune responses. Altogether, we show that RANO increases the efficacy of targeted melanoma therapy through its effects on FAO and the methionine salvage pathway. Importantly, our study suggests that RANO could sensitize BRAFi-resistant tumours to immunotherapy. Since RANO has very mild side-effects, it might constitute a therapeutic option to improve the two main strategies currently used to treat metastatic melanoma., (© 2023. The Author(s).)

Recent studies highlight the importance of baseline functional immunity for immune checkpoint blockade therapies. High-dimensional systemic immune profiling is performed in a cohort of non-small-cell lung cancer patients undergoing PD-L1/PD-1 blockade immunotherapy. Responders show high baseline myeloid phenotypic diversity in peripheral blood. To quantify it, we define a diversity index as a potential biomarker of response. This parameter correlates with elevated activated monocytic cells and decreased granulocytic phenotypes. High-throughput profiling of soluble factors in plasma identifies fractalkine (FKN), a chemokine involved in immune chemotaxis and adhesion, as a biomarker of response to immunotherapy that also correlates with myeloid cell diversity in human patients and murine models. Secreted FKN inhibits lung adenocarcinoma growth in vivo through a prominent contribution of systemic effector NK cells and increased tumor immune infiltration. FKN sensitizes murine lung cancer models refractory to anti-PD-1 treatment to immune checkpoint blockade immunotherapy. Importantly, recombinant FKN and tumor-expressed FKN are efficacious in delaying tumor growth in vivo locally and systemically, indicating a potential therapeutic use of FKN in combination with immunotherapy., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Bispecific antibodies are a promising type of therapy for the treatment of cancer due to their ability to simultaneously inhibit different proteins playing a role in cancer progression. The development in lung cancer has been singularly intense because of the increasingly vast knowledge of the underlying molecular routes, in particular, in oncogene-driven tumors. In this review, we present the current landscape of bispecific antibodies for the treatment of lung cancer and discuss potential scenarios where the role of these therapeutics might expand in the near future.

The application of BNT162b2 and mRNA-1273 vaccines against SARS-CoV-2 infection has constituted a determinant resource to control the COVID-19 pandemic. Since the beginning of 2021, millions of doses have been administered in several countries of North and South America and Europe. Many studies have confirmed the efficacy of these vaccines in a wide range of ages and in vulnerable groups of people against COVID-19. Nevertheless, the emergence and selection of new variants have led to a progressive decay in vaccine efficacy. Pfizer-BioNTech and Moderna developed updated bivalent vaccines-Comirnaty and Spikevax-to improve responses against the SARS-CoV-2 Omicron variants. Frequent booster doses with monovalent or bivalent mRNA vaccines, the emergence of some rare but serious adverse events and the activation of T-helper 17 responses suggest the need for improved mRNA vaccine formulations or the use of other types of vaccines. In this review, we discuss the advantages and limitations of mRNA vaccines targeting SARS-CoV-2 focusing on the most recent, related publications.

Immune-checkpoint inhibitors (ICIs) are antagonists of inhibitory receptors in the immune system, such as the cytotoxic T-lymphocyte-associated antigen-4, the programmed cell death protein-1 and its ligand PD-L1, and they are increasingly used in cancer treatment. By blocking certain suppressive pathways, ICIs promote T-cell activation and antitumor activity but may induce so-called immune-related adverse events (irAEs), which mimic traditional autoimmune disorders. With the approval of more ICIs, irAE prediction has become a key factor in improving patient survival and quality of life. Several biomarkers have been described as potential irAE predictors, some of them are already available for clinical use and others are under development; examples include circulating blood cell counts and ratios, T-cell expansion and diversification, cytokines, autoantibodies and autoantigens, serum and other biological fluid proteins, human leucocyte antigen genotypes, genetic variations and gene profiles, microRNAs, and the gastrointestinal microbiome. Nevertheless, it is difficult to generalize the application of irAE biomarkers based on the current evidence because most studies have been retrospective, time-limited and restricted to a specific type of cancer, irAE or ICI. Long-term prospective cohorts and real-life studies are needed to assess the predictive capacity of different potential irAE biomarkers, regardless of the ICI type, organ involved or cancer site.

Immunotherapies based on immune checkpoint blockade have shown remarkable clinical outcomes and durable responses in patients with many tumor types. Nevertheless, these therapies lack efficacy in most cancer patients, even causing severe adverse events in a small subset of patients, such as inflammatory disorders and hyper-progressive disease. To diminish the risk of developing serious toxicities, intratumor delivery of monoclonal antibodies could be a solution. Encouraging results have been shown in both preclinical and clinical studies. Thus, intratumor immunotherapy as a new strategy may retain efficacy while increasing safety. This approach is still an exploratory frontier in cancer research and opens up new possibilities for next-generation personalized medicine. Local intratumor delivery can be achieved through many means, but an attractive approach is the use of gene therapy vectors expressing mAbs inside the tumor mass. Here, we summarize basic, translational, and clinical results of intratumor mAb delivery, together with descriptions of non-viral and viral strategies for mAb delivery in preclinical and clinical development. Currently, this is an expanding research subject that will surely play a key role in the future of oncology.

Chimeric antigen receptor (CAR)-T adoptive cell therapy is one of the most promising advanced therapies for the treatment of cancer, with unprecedented outcomes in haematological malignancies. However, it still lacks efficacy in solid tumours, possibly because engineered T cells become inactive within the immunosuppressive tumour microenvironment (TME). In the TME, cells of the myeloid lineage (M) are among the immunosuppressive cell types with the highest tumour infiltration rate. These cells interact with other immune cells, mediating immunosuppression and promoting angiogenesis. Recently, the development of CAR-M cell therapies has been put forward as a new candidate immunotherapy with good efficacy potential. This alternative CAR strategy may increase the efficacy, survival, persistence, and safety of CAR treatments in solid tumours. This remains a critical frontier in cancer research and opens up a new possibility for next-generation personalised medicine to overcome TME resistance. However, the exact mechanisms of action of CAR-M and their effect on the TME remain poorly understood. Here, we summarise the basic, translational, and clinical results of CAR-innate immune cells and CAR-M cell immunotherapies, from their engineering and mechanistic studies to preclinical and clinical development.

PD-L1/PD-1 blockade immunotherapy has changed the therapeutic approaches for the treatment of many cancers. Nevertheless, the mechanisms underlying its efficacy or treatment failure are still unclear. Proficient systemic immunity seems to be a prerequisite for efficacy, as recently shown in patients and in mouse models. It is widely accepted that expansion of anti-tumor CD8 T cell populations is principally responsible for anti-tumor responses. In contrast, the role of CD4 T cells has been less studied. Here we review and discuss the evidence supporting the contribution of CD4 T cells to anti-tumor immunity, especially recent advances linking CD4 T cell subsets to efficacious PD-L1/PD-1 blockade immunotherapy. We also discuss the role of CD4 T cell memory subsets present in peripheral blood before the start of immunotherapies, and their utility as predictors of response.

It is unclear whether patients with cancer present inherently impaired responses to COVID-19 and vaccination due to their treatments, neoplastic diseases or both. To address this question, immune profiling was performed in three cohorts of healthy donors and oncologic patients: infected with SARS-CoV-2, BNT162b2-vaccinated, and with previous COVID-19 disease and subsequently vaccinated. Cancer patients showed good antibody responses to vaccination, but poor induction of T-cell responses towards the S protein when compared to infection. Following natural infection, the major targets for T-cells were the SARS-CoV-2 structural proteins M and S, but not the N protein. Similar to antibody titers, the T-cell responses quickly decayed after six months post-vaccination. Significant memory T-cell expansion was observed in vaccinated donors only if previously diagnosed with COVID-19 before undergoing vaccination. Oncologic patients with previous COVID-19 followed by vaccination exhibited potent IL-17+ CD4 and CD8 T-cell responses and elevated numbers of circulating neutrophils in peripheral blood.

One of the most common symptoms in COVID-19 is a sudden loss of smell. SARS-CoV-2 has been detected in the olfactory bulb (OB) from animal models and sporadically in COVID-19 patients. To decipher the specific role over the SARS-CoV-2 proteome at olfactory level, we characterized the in-depth molecular imbalance induced by the expression of GFP-tagged SARS-CoV-2 structural proteins (M, N, E, S) on mouse OB cells. Transcriptomic and proteomic trajectories uncovered a widespread metabolic remodeling commonly converging in extracellular matrix organization, lipid metabolism and signaling by receptor tyrosine kinases. The molecular singularities and specific interactome expression modules were also characterized for each viral structural factor. The intracellular molecular imbalance induced by each SARS-CoV-2 structural protein was accompanied by differential activation dynamics in survival and immunological routes in parallel with a differentiated secretion profile of chemokines in OB cells. Machine learning through a proteotranscriptomic data integration uncovered TGF-beta signaling as a confluent activation node by the SARS-CoV-2 structural proteome. Taken together, these data provide important avenues for understanding the multifunctional immunomodulatory properties of SARS-CoV-2 M, N, S and E proteins beyond their intrinsic role in virion formation, deciphering mechanistic clues to the olfactory inflammation observed in COVID-19 patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lachén-Montes, Mendizuri, Ausín, Echaide, Blanco, Chocarro, de Toro, Escors, Fernández-Irigoyen, Kochan and Santamaría.)

Vaccination, being able to prevent millions of cases of infectious diseases around the world every year, is the most effective medical intervention ever introduced. However, immunosenescence makes vaccines less effective in providing protection to older people. Although most studies explain that this is mainly due to the immunosenescence of T and B cells, the immunosenescence of innate immunity can also be a significant contributing factor. Alterations in function, number, subset, and distribution of blood neutrophils, monocytes, and natural killer and dendritic cells are detected in aging, thus potentially reducing the efficacy of vaccines in older individuals. In this paper, we focus on the immunosenescence of the innate blood immune cells. We discuss possible strategies to counteract the immunosenescence of innate immunity in order to improve the response to vaccination. In particular, we focus on advances in understanding the role and the development of new adjuvants, such as TLR agonists, considered a promising strategy to increase vaccination efficiency in older individuals.

The number of people that are 65 years old or older has been increasing due to the improvement in medicine and public health. However, this trend is not accompanied by an increase in quality of life, and this population is vulnerable to most illnesses, especially to infectious diseases. Vaccination is the best strategy to prevent this fact, but older people present a less efficient response, as their immune system is weaker due mainly to a phenomenon known as immunosenescence. The adaptive immune system is constituted by two types of lymphocytes, T and B cells, and the function and fitness of these cell populations are affected during ageing. Here, we review the impact of ageing on T and B cells and discuss the approaches that have been described or proposed to modulate and reverse the decline of the ageing adaptive immune system.

Single-agent immunotherapy has been widely accepted as frontline treatment for advanced non-small cell lung cancer (NSCLC) with high tumor PD-L1 expression, but most patients do not respond and the mechanisms of resistance are not well known. Several works have highlighted the immunosuppressive activities of myeloid subpopulations, including low-density neutrophils (LDNs), although the context in which these cells play their role is not well defined. We prospectively monitored LDNs in peripheral blood from patients with NSCLC treated with anti-PD-1 immune checkpoint inhibitors (ICIs) as frontline therapy, in a cohort of patients treated with anti-PD1 immunotherapy combined with chemotherapy (CT+IT), and correlated values with outcomes. We explored the underlying mechanisms through ex vivo experiments. Elevated baseline LDNs predict primary resistance to ICI monotherapy in patients with NSCLC, and are not associated with response to CT+IT. Circulating LDNs mediate resistance in NSCLC receiving ICI as frontline therapy through humoral immunosuppression. A depletion of this population with CT+IT might overcome resistance, suggesting that patients with high PD-L1 tumor expression and high baseline LDNs might benefit from this combination. The activation of the HGF/c-MET pathway in patients with elevated LDNs revealed by quantitative proteomics supports potential drug combinations targeting this pathway.

Immune checkpoint inhibitors (ICIs) have revolutionized medical practice in oncology since the FDA approval of the first ICI 11 years ago. In light of this, Lymphocyte-Activation Gene 3 (LAG-3) is one of the most important next-generation immune checkpoint molecules, playing a similar role as Programmed cell Death protein 1 (PD-1) and Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4). 19 LAG-3 targeting molecules are being evaluated at 108 clinical trials which are demonstrating positive results, including promising bispecific molecules targeting LAG-3 simultaneously with other ICIs. Recently, a new dual anti-PD-1 (Nivolumab) and anti-LAG-3 (Relatimab) treatment developed by Bristol Myers Squibb (Opdualag), was approved by the Food and Drug Administration (FDA) as the first LAG-3 blocking antibody combination for unresectable or metastatic melanoma. This novel immunotherapy combination more than doubled median progression-free survival (PFS) when compared to nivolumab monotherapy (10.1 months versus 4.6 months). Here, we analyze the large clinical trial responsible for this historical approval (RELATIVITY-047), and discuss the preclinical and clinical developments that led to its jump into clinical practice. We will also summarize results achieved by other LAG-3 targeting molecules with promising anti-tumor activities currently under clinical development in phases I, I/II, II, and III. Opdualag will boost the entry of more LAG-3 targeting molecules into clinical practice, supporting the accumulating evidence highlighting the pivotal role of LAG-3 in cancer.

Background: Improving cancer immunotherapy long-term clinical benefit is a major priority. It has become apparent that multiple axes of immune suppression restrain the capacity of T cells to provide anti-tumour activity including signalling through PD1/PD-L1 and LAG3/MHC-II., Methods: CB213 has been developed as a fully human PD1/LAG3 co-targeting multi-specific Humabody composed of linked V H domains that avidly bind and block PD1 and LAG3 on dual-positive T cells. We present the preclinical primary pharmacology of CB213: biochemistry, cell-based function vs. immune-suppressive targets, induction of T cell proliferation ex vivo using blood obtained from NSCLC patients, and syngeneic mouse model anti-tumour activity. CB213 pharmacokinetics was assessed in cynomolgus macaques., Results: CB213 shows picomolar avidity when simultaneously engaging PD1 and LAG3. Assessing LAG3/MHC-II or PD1/PD-L1 suppression individually, CB213 preferentially counters the LAG3 axis. CB213 showed superior activity vs. αPD1 antibody to induce ex vivo NSCLC patient T cell proliferation and to suppress tumour growth in a syngeneic mouse tumour model, for which both experimental systems possess PD1 and LAG3 suppressive components. Non-human primate PK of CB213 suggests weekly clinical administration., Conclusions: CB213 is poised to enter clinical development and, through intercepting both PD1 and LAG3 resistance mechanisms, may benefit patients with tumours escaping front-line immunological control., (© 2021. The Author(s).)

Immune checkpoint blockade (ICB) of the PD-1 pathway revolutionized the survival forecast for advanced non-small cell lung cancer (NSCLC). Yet, the majority of PD-L1 + NSCLC patients are refractory to anti-PD-L1 therapy. Recent observations indicate a pivotal role for the PD-L1 + tumor-infiltrating myeloid cells in therapy failure. As the latter comprise a heterogenous population in the lung tumor microenvironment, we applied an orthotopic Lewis Lung Carcinoma (LLC) model to evaluate 11 different tumor-residing myeloid subsets in response to anti-PD-L1 therapy. While we observed significantly reduced fractions of tumor-infiltrating MHC-II low macrophages and monocytes, serological levels of TNF-α restored in lung tumor-bearing mice. Notably, we demonstrated in vivo and in vitro that anti-PD-L1 therapy mediated a monocyte-specific production of, and response to TNF-α, further accompanied by their significant upregulation of CD80, VISTA, LAG-3, SIRP-α and TIM-3. Nevertheless, co-blockade of PD-L1 and TNF-α did not reduce LLC tumor growth. A phenomenon that was partly explained by the observation that monocytes and TNF-α play a Janus-faced role in anti-PD-L1 therapy-mediated CTL stimulation. This was endorsed by the observation that monocytes appeared crucial to effectively boost T cell-mediated LLC killing in vitro upon combined PD-L1 with LAG-3 or SIRP-α blockade. Hence, this study enlightens the biomarker potential of lung tumor-infiltrated monocytes to define more effective ICB combination strategies., Competing Interests: MK, KB, and QL have a granted patent on “Human PD-L1 binding immunoglobulins” (WO2019166622A1). MK has patents on the use of nanobodies for imaging and therapy. MK received research funding from Precirix. MK has ownership in AbScint which leverages nanobody imaging tracers into clinical application. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 De Ridder, Locy, Piccioni, Zuazo, Awad, Verhulst, Van Bulck, De Vlaeminck, Lecocq, Reijmen, De Mey, De Beck, Ertveldt, Pintelon, Timmermans, Escors, Keyaerts, Breckpot and Goyvaerts.)

Adoptive cell therapy with genetically modified T lymphocytes that express chimeric antigen receptors (CAR-T) is one of the most promising advanced therapies for the treatment of cancer, with unprecedented outcomes in hematological malignancies. However, the efficacy of CAR-T cells in solid tumors is still very unsatisfactory, because of the strong immunosuppressive tumor microenvironment that hinders immune responses. The development of next-generation personalized CAR-T cells against solid tumors is a clinical necessity. The identification of therapeutic targets for new CAR-T therapies to increase the efficacy, survival, persistence, and safety in solid tumors remains a critical frontier in cancer immunotherapy. Here, we summarize basic, translational, and clinical results of CAR-T cell immunotherapies in lung cancer, from their molecular engineering and mechanistic studies to preclinical and clinical development.

Myeloid-derived suppressor cells (MDSCs) play a major role in cancer progression. In this study, we investigated the mechanisms by which complement C5a increases the capacity of polymorphonuclear MDSCs (PMN-MDSCs) to promote tumor growth and metastatic spread. Stimulation of PMN-MDSCs with C5a favored the invasion of cancer cells via a process dependent on the formation of neutrophil extracellular traps (NETs). NETosis was dependent on the production of high mobility group box 1 (HMGB1) by cancer cells. Moreover, C5a induced the surface expression of the HMGB1 receptors TLR4 and RAGE in PMN-MDSCs. In a mouse lung metastasis model, inhibition of C5a, C5a receptor-1 (C5aR1) or NETosis reduced the number of circulating-tumor cells (CTCs) and the metastatic burden. In support of the translational relevance of these findings, C5a was able to stimulate migration and NETosis in PMN-MDSCs obtained from lung cancer patients. Furthermore, myeloperoxidase (MPO)-DNA complexes, as markers of NETosis, were elevated in lung cancer patients and significantly correlated with C5a levels. In conclusion, C5a induces the formation of NETs from PMN-MDSCs in the presence of cancer cells, which may facilitate cancer cell dissemination and metastasis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Monocytic and granulocytic myeloid-derived suppressor cells together with tumor-infiltrating macrophages constitute the main tumor-infiltrating immunosuppressive myeloid populations. Due to the phenotypic resemblance to conventional myeloid cells, their identification and purification from within the tumors is technically difficult and makes their study a challenge. We differentiated myeloid cells modeling the three main tumor-infiltrating types together with uncommitted macrophages, using ex vivo differentiation methods resembling the tumor microenvironment. The phenotype and proteome of these cells was compared to identify linage-dependent relationships and cancer-specific interactome expression modules. The relationships between monocytic MDSCs and TAMs, monocytic MDSCs and granulocytic MDSCs, and hierarchical relationships of expression networks and transcription factors due to lineage and cancer polarization were mapped. Highly purified immunosuppressive myeloid cell populations that model tumor-infiltrating counterparts were systematically analyzed by quantitative proteomics. Full functional interactome maps have been generated to characterize at high resolution the relationships between the three main myeloid tumor-infiltrating cell types. Our data highlights the biological processes related to each cell type, and uncover novel shared and differential molecular targets. Moreover, the high numbers and fidelity of ex vivo-generated subsets to their natural tumor-shaped counterparts enable their use for validation of new treatments in high-throughput experiments.

Lymphocyte activation gene 3 (LAG-3) is a cell surface inhibitory receptor with multiple biological activities over T cell activation and effector functions. LAG-3 plays a regulatory role in immunity and emerged some time ago as an inhibitory immune checkpoint molecule comparable to PD-1 and CTLA-4 and a potential target for enhancing anti-cancer immune responses. LAG-3 is the third inhibitory receptor to be exploited in human anti-cancer immunotherapies, and it is considered a potential next-generation cancer immunotherapy target in human therapy, right next to PD-1 and CTLA-4. Unlike PD-1 and CTLA-4, the exact mechanisms of action of LAG-3 and its relationship with other immune checkpoint molecules remain poorly understood. This is partly caused by the presence of non-conventional signaling motifs in its intracellular domain that are different from other conventional immunoregulatory signaling motifs but with similar inhibitory activities. Here we summarize the current understanding of LAG-3 signaling and its role in LAG-3 functions, from its mechanisms of action to clinical applications.

Along with the positioning of immunotherapy as a preferential treatment for a wide variety of neoplasms, a new pattern of response consisting in a sudden acceleration of tumor growth has been described. This phenomenon has received the name of "hyperprogressive disease", and several definitions have been proposed for its identification, most of them relying on radiological criteria. However, due to the fact that the cellular and molecular mechanisms have not been elucidated yet, there is still some debate regarding whether this fast progression is induced by immunotherapy or only reflects the natural course of some highly aggressive neoplasms. Moreover, contradictory results of trials including patients with different cancer types suggest that both the incidence, the associated factors and the implications regarding prognosis might differ depending on tumor histology. This article intends to review the main publications regarding this matter and critically approach the most controversial aspects.

PD-L1/PD-1 blockade immunotherapy has significantly improved treatment outcome for several cancer types compared to conventional cytotoxic therapies. However, the specific molecular and cellular mechanisms behind its efficacy are currently unclear. There is increasing evidence in murine models and in patients that unveil the key importance of systemic immunity to achieve clinical responses under several types of immunotherapy. Indeed, PD-L1/PD-1 blockade induces the expansion of systemic CD8+ PD-1+ T cell subpopulations which might be responsible for direct anti-tumor responses. However, the role of CD4+ T cells in PD-L1/PD-1 blockade-induced anti-tumor responses has been less documented. In this review we focus on the experimental data supporting the "often suspected" indispensable helper function of CD4 T cells towards CD8 effector anti-tumor responses in cancer; and particularly, we highlight the recently published studies uncovering the key contribution of systemic CD4 T cells to clinical efficacy in PD-L1/PD-1 blockade therapies. We conclude and propose that the presence of specific CD4 T cell memory subsets in peripheral blood before the initiation of treatments is a strong predictor of responses in non-small cell lung cancer patients. Therefore, development of new approaches to improve CD4 responses before PD-L1/PD-1 blockade therapy could be the solution to increase response rates and survival of patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Zuazo, Arasanz, Bocanegra, Fernandez, Chocarro, Vera, Kochan and Escors.)

The search for non-invasive systemic biomarkers of response to PD-L1/PD-1 blockade immunotherapy is currently a priority in oncoimmunology. In contrast to classical tumor biomarkers, the identification of clinically useful immunological biomarkers is certainly a challenge, as anti-cancer immune responses depend on the coordinated action of many cell types. Studies on the dynamics of systemic CD8 T-cell populations have provided indications that such biomarkers may have a place in clinical practice. However, the power of CD8 T-cell subsets to discriminate clinical responses in immunotherapy has so far proven to be limited. The systemic evaluation of CD8 T-cell regulators such as myeloid cells and CD4 T cells may provide the solution. Here we discuss the value of systemic quantification of CD4 T-cell subsets for patient selection in light of the results obtained by Prof. Kagamu's and our team. Our studies have independently demonstrated that the evaluation of the pre-treatment status of systemic CD4 immunity is a critical factor for the clinical outcome of PD-L1/PD-1 blockade therapy with robust predictive capacities., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

The use of monoclonal antibodies targeting PD-1/PD-L1 axis completely changed anticancer treatment strategies. However, despite the significant improvement in overall survival and progression-free survival of patients undergoing these immunotherapy treatments, the only clinically accepted biomarker with some prediction capabilities for the outcome of the treatment is PD-L1 expression in tumor biopsies. Nevertheless, even when having PD-L1-positive tumors, numerous patients do not respond to these treatments. Considering the high cost of these therapies and the risk of immune-related adverse events during therapy, it is necessary to identify additional biomarkers that would facilitate stratifying patients in potential responders and non-responders before the start of immunotherapies. Here, we review the utility of PD-L1 expression not only in tumor cells but in immune system cells and their influence on the antitumor activity of immune cell subsets.

Cancer cells acquire resistance to cytotoxic therapies targeting major survival pathways by adapting their metabolism. The AKT pathway is a major regulator of human pancreatic adenocarcinoma progression and a key pharmacological target. The mechanisms of adaptation to long-term silencing of AKT isoforms of human and mouse pancreatic adenocarcinoma cancer cells were studied. Following silencing, cancer cells remained quiescent for long periods of time, after which they recovered proliferative capacities. Adaptation caused profound proteomic changes largely affecting mitochondrial biogenesis, energy metabolism and acquisition of a number of distinct cancer stem cell (CSC) characteristics depending on the AKT isoform that was silenced. The adaptation to AKT1 silencing drove most de-differentiation and acquisition of stemness through C-MYC down-modulation and NANOG upregulation, which were required for survival of adapted CSCs. The changes associated to adaptation sensitized cancer cells to inhibitors targeting regulators of oxidative respiration and mitochondrial biogenesis. In vivo pharmacological co-inhibition of AKT and mitochondrial metabolism effectively controlled pancreatic adenocarcinoma growth in pre-clinical models.

Cancer immunotherapies targeting immune checkpoints such as programmed cell-death protein 1 (PD-1) and its ligand programmed cell-death 1 ligand 1 (PD-L1), are revolutionizing cancer treatment and transforming the practice of medical oncology. However, despite all the recent successes of this type of immunotherapies, most patients are still refractory and present either intrinsic resistance or acquired resistance. Either way, this is a major clinical problem and one of the most significant challenges in oncology. Therefore, the identification of biomarkers to predict clinical responses or for patient stratification by probability of response has become a clinical necessity. However, the mechanisms leading to PD-L1/PD-1 blockade resistance are still poorly understood. A deeper understanding of the basic mechanisms underlying resistance to cancer immunotherapies will provide insight for further development of novel strategies designed to overcome resistance and treatment failure. Here we discuss some of the major molecular mechanisms of resistance to PD-L1/PD-1 immune checkpoint blockade and argue whether tumor intrinsic or extrinsic factors constitute main determinants of response and resistance., (Copyright © 2020 Chocarro de Erauso, Zuazo, Arasanz, Bocanegra, Hernandez, Fernandez, Garcia-Granda, Blanco, Vera, Kochan and Escors.)

The development of cancer immunotherapy in the last decade has followed a vertiginous rhythm. Nowadays, immune checkpoint inhibitors (ICI) which include anti-CTLA4, anti-PD-1 and anti-PD-L1 antibodies are in clinical use for the treatment of numerous cancers. However, approximately only a third of the patients benefit from ICI therapies. Many efforts have been made for the identification of biomarkers allowing patient stratification into potential responders and progressors before the start of ICI therapies or for monitoring responses during treatment. While much attention is centered on biomarkers from the tumor microenvironment, in many cases biopsies are not available. The identification of systemic immune cell subsets that correlate with responses could provide promising biomarkers. Some of them have been reported to influence the response to ICI therapies, such as proliferation and activation status of CD8 and CD4 T cells, the expression of immune checkpoints in peripheral blood cells and the relative numbers of immunosuppressive cells such as regulatory T cells and myeloid-derived suppressor cells. In addition, the profile of soluble factors in plasma samples could be associated to response or tumor progression. Here we will review the cellular subsets associated to response or progression in different studies and discuss their accuracy in diagnosis.