The role of metabolism in tumor immune evasion: Novel approaches to improve immunotherapy

The tumor microenvironment exhibits altered metabolic properties as a consequence of the needs of tumor cells, the natural selection of the most adapted clones, and the selfish relationship with other cell types. Beyond its role in supporting uncontrolled tumor growth, through energy and building materials obtention, metabolism is a key element controlling tumor immune evasion. Immunotherapy has revolutionized the treatment of cancer, being the first line of treatment for multiple types of malignancies. However, many patients either do not benefit from immunotherapy or eventually relapse. In this review we overview the immunoediting process with a focus on the metabolism-related elements that are responsible for increased immune evasion, either through reduced immunogenicity or increased resistance of tumor cells to the apoptotic action of immune cells. Finally, we describe the main molecules to modulate these immune evasion processes through the control of the metabolic microenvironment as well as their clinical developmental statusWork in the authors’ laboratories was supported by “Instituto de Salud Carlos III” (ISCIII) PI19/01652 grant, Ministry of Science and Innovation RTC2017-6502-1 INmunoSIGHT and European Union’s Horizon 2020 research and innovation programme, CLARIFY 875160 grant, to M.P. A.C.-B. received a Spanish Lung Cancer Group (SLCG) grant and is supported by a ISCIII-“Sara Borrell” contract CD19/00170. M.C. is supported by PEJD-2019-PRE/BMD-17006 contract granted to A.C.-B. R.L.-B. was supported by PEJ16/MED/AI-1972 and PEJD-2018-PRE/SAL-8641 from European Social Fund and Comunidad de Madrid, both granted to M.

Clinical and molecular parameters associated to pneumonitis development in non-small-cell lung cancer patients receiving chemoimmunotherapy from NADIM trial

Background Pneumonitis (Pn) is one of the main immune-related adverse effects, having a special importance in lung cancer, since they share affected tissue. Despite its clinical relevance, Pn development remains an unpredictable treatment adverse effect, whose mechanisms are mainly unknown, being even more obscure when it is associated to chemoimmunotherapy.Methods In order to identify parameters associated to treatment related Pn, we analyzed clinical variables and molecular parameters from 46 patients with potentially resectable stage IIIA non-small-cell lung cancer treated with neoadjuvant chemoimmunotherapy included in the NADIM clinical trial (NCT03081689). Pn was defined as clinical or radiographic evidence of lung inflammation without alternative diagnoses, from treatment initiation to 180 days. Results Among 46 patients, 12 developed Pn (26.1%). Sex, age, smoking status, packs-year, histological subtype, clinical or pathological response, progression free survival, overall survival and number of nivolumab cycles, were not associated to Pn development. Regarding molecular parameters at diagnosis, Pn evelopment was not associated to programmed death ligand 1, TPS, T cell receptor repertoire parameters, or tumor mutational burden. However, patients who developed Pn had statistically significant lower blood median levels of platelet to monocyte ratio (p=0.012) and teratocarcinoma derived growth factor 1 (p=0.013; area under the curve (AUC) 0.801), but higher median percentages of natural killers (NKs) (p=0.019; AUC 0.786), monocytes (p=0.017; AUC 0.791), MSP (p=0.006; AUC 0.838), PARN (p=0.017; AUC 0.790), and E-Cadherin (p=0.022; AUC 0.788). In addition, the immune scenario of Pn after neoadjuvant treatment involves: high levels of neutrophils and NK cells, but low levels of B and T cells in peripheral blood; increased clonality of intratumoral T cells; and elevated plasma levels of several growth factors (EGF, HGF, VEGF, ANG-1, PDGF, NGF, and NT4) and inflammatory cytokines (MIF, CCL16, neutrophil gelatinase-associated lipocalin, BMP-4, and u-PAR). Conclusions Although statistically underpowered, our results shed light on the possible mechanisms behind Pn development, involving innate and adaptative immunity, and open the possibility to predict patients at high risk. If confirmed, this may allow the personalization of both, the surveillance strategy and the therapeutic approaches to manage Pn in patients receiving chemoimmunotherapy

Evaluation of cell culture in microfluidic chips for application in monoclonal antibody production

Microfluidic chips are useful devices for cell culture that allow cell growth under highly controlled conditions, as is required for production of therapeutic recombinant proteins. To understand the optimal conditions for growth of cells amenable of recombinant protein expression in these devices,we culturedHEK-293T cells under different microfluidic experimental conditions. The cells were cultured in polymethyl methacrylate (PMMA) and polydi-methylsiloxane (PDMS)microdevices, in the absence or presence of the cell adhesion agent poly-D-lysine. Different microchannel geometries and thicknesses, as well as the influence of the flow rate have also been tested, showing their great influence in cell adhesion and growth. Results show that the presence of poly-D-lysine improves the adhesion and viability of the cells in continuous or discontinuous flow. Moreover, the optimal adhesion of cells was observed in the corners of themicrochannels, as well as in wide channels possibly due to the decrease in the flow rate in these areas. These studies provide insight into the optimal architecture of microchannels for long-term culture of adherent cells in order to use microfluidics devices as bioreactors for monoclonal antibodies production.Fil: Peñaherrera Pazmiño, Ana Belén. Universidad Tecnológica Nacional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Payés, Cristian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Sierra Rodero, Marina. Universidad Tecnológica Nacional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vega, M.. Universidad Tecnológica Nacional; ArgentinaFil: Rosero, G.. Universidad Tecnológica Nacional; Argentina. Universidad de Buenos Aires; ArgentinaFil: Lerner, Betiana. Universidad Tecnológica Nacional; Argentina. Universidad de Buenos Aires; ArgentinaFil: Helguera, Gustavo Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Pérez, M. S.. Universidad Tecnológica Nacional; Argentina. Universidad de Buenos Aires; Argentin

Tumor microenvironment gene expression profiles associated to complete pathological response and disease progression in resectable NSCLC patients treated with neoadjuvant chemoimmunotherapy

Background Neoadjuvant chemoimmunotherapy for non-small cell lung cancer (NSCLC) has improved pathological responses and survival rates compared with chemotherapy alone, leading to Food and Drug Administration (FDA) approval of nivolumab plus chemotherapy for resectable stage IB-IIIA NSCLC (AJCC 7th edition) without ALK or EGFR alterations. Unfortunately, a considerable percentage of tumors do not completely respond to therapy, which has been associated with early disease progression. So far, it is impossible to predict these events due to lack of knowledge. In this study, we characterized the gene expression profile of tumor samples to identify new biomarkers and mechanisms behind tumor responses to neoadjuvant chemoimmunotherapy and disease recurrence after surgery. Methods Tumor bulk RNA sequencing was performed in 16 pretreatment and 36 post-treatment tissue samples from 41 patients with resectable stage IIIA NSCLC treated with neoadjuvant chemoimmunotherapy from NADIM trial. A panel targeting 395 genes related to immunological processes was used. Tumors were classified as complete pathological response (CPR) and non-CPR, based on the total absence of viable tumor cells in tumor bed and lymph nodes tested at surgery. Differential-expressed genes between groups and pathway enrichment analysis were assessed using DESeq2 and gene set enrichment analysis. CIBERSORTx was used to estimate the proportions of immune cell subtypes. Results CPR tumors had a stronger pre-established immune infiltrate at baseline than non-CPR, characterized by higher levels of IFNG, GZMB, NKG7, and M1 macrophages, all with a significant area under the receiver operating characteristic curve (ROC) >0.9 for CPR prediction. A greater effect of neoadjuvant therapy was also seen in CPR tumors with a reduction of tumor markers and IFN gamma signaling after treatment. Additionally, the higher expression of several genes, including AKT1, BST2, OAS3, or CD8B; or higher dendritic cells and neutrophils proportions in post-treatment non-CPR samples, were associated with relapse after surgery. Also, high pretreatment PD-L1 and tumor mutational burden levels influenced the post-treatment immune landscape with the downregulation of proliferation markers and type I interferon signaling molecules in surgery samples. Conclusions Our results reinforce the differences between CPR and non-CPR responses, describing possible response and relapse immune mechanisms, opening the possibility of therapy personalization of immunotherapy-based regimens in the neoadjuvant setting of NSCLC

Perioperative Chemoimmunotherapy Induces Strong Immune Responses and Long-Term Survival in Patients With Hla Class I-deficient Non-small Cell Lung Cancer

BACKGROUND: Loss of human leukocyte antigen (HLA) class I expression and loss of heterozygosity (LOH) are common events implicated in the primary resistance of non-small cell lung cancer (NSCLC) to immunotherapy. However, there is no data on perioperative chemoimmunotherapy (ChIO) efficacy or response mechanisms in the context of HLA class I defects. METHODS: Baseline HLA class I tumor status (HLA-deficient (HLA-DEF) or HLA-proficient (HLA-PRO)) was determined by DNA LOH combined with immunohistochemistry for protein levels in tissue of 24 patients with NSCLC treated with perioperative nivolumab plus chemotherapy from NADIM trial (NCT03081689). We integrated HLA tumor status with molecular data (programmed death-ligand 1 (PD-L1), TMB, TCR repertoire, TILs populations, bulk RNA-seq, and spatial transcriptomics (ST)) and clinical outcomes (pathological response and survival data) to study the activity of perioperative ChIO considering HLA class I defects. RESULTS: HLA-DEF tumors comprised 41.7% of analyzed tumors and showed a desert-like microenvironment at baseline, with lower PD-L1 levels and reduced immune infiltrate. However, perioperative ChIO induced similar complete pathological response (CPR) rates in both HLA-DEF and PRO tumors (50% and 60% respectively, p=0.670), as well as 3-year survival rates: Progression-free survival (PFS) and overall survival (OS) of 70% (95% CI 32.9% to 89.2%) for HLA-DEF, and PFS 71.4% (95% CI 40.6% to 88.2%) and OS 92.9% (95% CI 59.1% to 99.0%) for HLA-PRO (log-rank PFS p=0.909, OS p=0.137). Proof-of-concept ST analysis of a CPR HLA-DEF tumor after ChIO showed a strong immune response with tertiary lymphoid structures (TLS), CD4+T cells with HLA class II colocalization, and activated CD8+T cells. CONCLUSIONS: Our findings highlight the activity of perioperative ChIO, and the potential role of TLS and T-cell immune response, in NSCLC HLA-DEF tumors

Perioperative chemoimmunotherapy induces strong immune responses and long-term survival in patients with HLA class I-deficient non-small cell lung cancer

Background Loss of human leukocyte antigen (HLA) class I expression and loss of heterozygosity (LOH) are common events implicated in the primary resistance of non-small cell lung cancer (NSCLC) to immunotherapy. However, there is no data on perioperative chemoimmunotherapy (ChIO) efficacy or response mechanisms in the context of HLA class I defects.Methods Baseline HLA class I tumor status (HLA-deficient (HLA-DEF) or HLA-proficient (HLA-PRO)) was determined by DNA LOH combined with immunohistochemistry for protein levels in tissue of 24 patients with NSCLC treated with perioperative nivolumab plus chemotherapy from NADIM trial (NCT03081689). We integrated HLA tumor status with molecular data (programmed death-ligand 1 (PD-L1), TMB, TCR repertoire, TILs populations, bulk RNA-seq, and spatial transcriptomics (ST)) and clinical outcomes (pathological response and survival data) to study the activity of perioperative ChIO considering HLA class I defects.Results HLA-DEF tumors comprised 41.7% of analyzed tumors and showed a desert-like microenvironment at baseline, with lower PD-L1 levels and reduced immune infiltrate. However, perioperative ChIO induced similar complete pathological response (CPR) rates in both HLA-DEF and PRO tumors (50% and 60% respectively, p=0.670), as well as 3-year survival rates: Progression-free survival (PFS) and overall survival (OS) of 70% (95% CI 32.9% to 89.2%) for HLA-DEF, and PFS 71.4% (95% CI 40.6% to 88.2%) and OS 92.9% (95% CI 59.1% to 99.0%) for HLA-PRO (log-rank PFS p=0.909, OS p=0.137). Proof-of-concept ST analysis of a CPR HLA-DEF tumor after ChIO showed a strong immune response with tertiary lymphoid structures (TLS), CD4+T cells with HLA class II colocalization, and activated CD8+T cells.Conclusions Our findings highlight the activity of perioperative ChIO, and the potential role of TLS and T-cell immune response, in NSCLC HLA-DEF tumors

COVID-19 outbreaks in a transmission control scenario: challenges posed by social and leisure activities, and for workers in vulnerable conditions, Spain, early summer 2020

Severe acute respiratory syndrome coronavirus 2 community-wide transmission declined in Spain by early May 2020, being replaced by outbreaks and sporadic cases. From mid-June to 2 August, excluding single household outbreaks, 673 outbreaks were notified nationally, 551 active (>6,200 cases) at the time. More than half of these outbreaks and cases coincided with: (i) social (family/friends’ gatherings or leisure venues) and (ii) occupational (mainly involving workers in vulnerable conditions) settings. Control measures were accordingly applied

Clinical and molecular parameters associated to pneumonitis development in non-small-cell lung cancer patients receiving chemoimmunotherapy from NADIM trial

Background Pneumonitis (Pn) is one of the main immune-related adverse effects, having a specialimportance in lung cancer, since they share affected tissue. Despite its clinical relevance, Pn development remains an unpredictable treatment adverse effect, whose mechanisms are mainly unknown, being even more obscure when it is associated to chemoimmunotherapy. Methods In order to identify parameters associated to treatment related Pn, we analyzed clinical variables and molecular parameters from 46 patients with potentially resectable stage IIIA non-small-cell lung cancer treated with neoadjuvant chemoimmunotherapy included in the NADIM clinical trial (NCT03081689). Pn was defined asclinical or radiographic evidence of lung inflammation without alternative diagnoses, from treatment initiation to 180 days. Results Among 46 patients, 12 developed Pn (26.1%). Sex, age, smoking status, packs-year, histological subtype, clinical or pathological response, progression- free survival, overall survival and number of nivolumab cycles, were not associated to Pn development. Regarding molecular parameters at diagnosis, Pn development was not associated to programmed death ligand 1,TPS, T cell receptor repertoire parameters, or tumor mutational burden. However, patients who developed Pn had statistically significant lower blood median levels ofplatelet to monocyte ratio (p=0.012) and teratocarcinoma-derived growth factor 1 (p=0.013; area under the curve (AUC) 0.801), but higher median percentages of natural killers (NKs) (p=0.019; AUC 0.786), monocytes (p=0.017; AUC 0.791), MSP (p=0.006; AUC 0.838), PARN (p=0.017; AUC 0.790), and E-Cadherin (p=0.022; AUC 0.788). In addition, the immune scenario of Pn after neoadjuvant treatment involves: high levels of neutrophils and NK cells, but low levels of B and T cells in peripheral blood increased clonality of intratumoral T cells; and elevated plasma levels of several growth factors (EGF, HGF, VEGF,ANG-1, PDGF, NGF, and NT4) and inflammatory cytokines (MIF, CCL16, neutrophil gelatinase-associated lipocalin, BMP-4, and u-PAR). Conclusions Although statistically underpowered, our results shed light on the possible mechanisms behind Pn development, involving innate and adaptative immunity, and open the possibility to predict patients at high risk. If confirmed, this may allow the personalization of both, the surveillance strategy and the therapeutic approaches tomanage Pn in patients receiving chemoimmunotherapy

Accessible and Cost-Effective Method of PDMS Microdevices Fabrication Using a Reusable Photopolymer Mold

This work describes a novel and cost-effective method of polydimethylsiloxane (PDMS) microchips fabrication by using a printing plate photopolymer called Flexcel as a master mold (Fmold). This method has demonstrated the ability to generate multiple devices from a single master, reaching a minimum channel size of 25 μm, structures height ranging from 53 to 1500 μm and achieving dimensions of 1270 × 2062 mm2, which are larger than those obtained by the known techniques to date. Scanning electron microscopy, atomic force microscopy, and profilometry techniques have been employed to characterize the Fmold and PDMS replicas. The results showed high replication fidelity of Fmold to the PDMS replica. Furthermore, it was proved the reusability of the Fmold. In our study, up to 50 PDMS replicas have been fabricated without apparent degradation of the mold. The feasibility of the resulting PDMS replica was effectively demonstrated using a microfluidic device for enhanced oil recovery analysis.Fil: Bourguignon, Natalia. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Olmos Carreno, Carol Maritza. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sierra Rodero, Marina. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Peñaherrera Pazmiño, Ana Belén. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Tecnológica Nacional. Facultad Regional Haedo; ArgentinaFil: Rosero Yánez, Gustavo Ivan. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pineda Ramos, Pedro Antonio. Universidad Tecnológica Nacional. Facultad Regional Haedo; ArgentinaFil: Vizuete, Karla. Universidad de Las Fuerzas Armadas; EcuadorFil: Arroyo, Alejandro Carlos. Universidad de Las Fuerzas Armadas; EcuadorFil: Cumbal Flores, Luis. Universidad de Las Fuerzas Armadas; EcuadorFil: Lasorsa, Carlos Alberto. Universidad Tecnológica Nacional. Facultad Regional Haedo; ArgentinaFil: Perez, Maximiliano Sebastian. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lerner, Betiana. Universidad Tecnológica Nacional. Facultad Regional Haedo; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin