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IL12-based local gene therapy of cancer constitutes an active area of clinical research using plasmids, mRNAs, and viral vectors. To improve antitumor effects, we have experimentally tested the combination of mRNA constructs encoding IL12 and IL18. Moreover, we have used a form of IL18 [decoy-resistant IL18 (DR-18)] which has preserved bioactivity but does not bind to the IL18 binding protein decoy receptor. Both cytokines dramatically synergize to induce IFNγ release from mouse splenocytes, and, if systemically cotransferred to the liver, they mediate lethal toxicity. However, if given intratumorally to B16OVA tumor-bearing mice, the combination attains efficacy against the directly treated tumor and moderate tumor-delaying activity on distant noninjected lesions. Cotreatment was conducive to the presence of more activated CD8+ T cells in the treated and noninjected tumors. In keeping with these findings, the efficacy of treatment was contingent on the integrity of CD8+ T cells and cDC1 dendritic cells in the treated mice. Furthermore, efficacy of IL12 plus DR-18 local mRNA coinjection against distant concomitant tumors could be enhanced upon combination with anti–PD-1 mAb systemic treatment, thus defining a feasible synergistic immunotherapy strategy.

The ability of conventional type-1 dendritic cells (cDC1) to cross-present tumor antigens to CD8+ T cells is critical for the induction of antitumor cytotoxic T lymphocytes. Mice that are constitutively deficient in cDC1 cells have been reported to fail to respond to immunotherapy strategies based on checkpoint inhibitors. However, further work is needed to clarify the precise time during immunotherapy treatment that cDC1 cells are required for the beneficial effect of treatment. Here, we used a refined XCR1-DTR-Venus transgenic mouse model to acutely deplete cDC1 cells and trace their behavior using intravital microscopy. Diphtheria toxin-mediated cDC1 depletion prior to immunotherapy treatment with anti-PD-1 and/or anti-CD137 immunostimulatory monoclonal antibodies (mAbs) completely ablated anti-tumor efficacy. The efficacy of adoptive T-cell therapy was also hampered by prior cDC1 depletion. After the onset of immunotherapy treatment, depletion of cDC1s only moderately reduced the therapeutic efficacy of anti-PD-1 and anti-CD137 mAbs. Intravital microscopy of liver-engrafted tumors revealed changes in the intratumoral behavior of cDC1 cells in mice receiving immunotherapy, and treatment with diphtheria toxin to deplete cDC1s impaired tumor T-cell infiltration and function. These results reveal that the functional integrity of the cDC1 compartment is required at the onset of various immunotherapies to successfully treat established tumors. This work was supported by Spanish Ministry of Economy and Competitiveness and Spanish Ministry of Research (MINECO SAF2014-52361-R and SAF 2017-83267-C2-1R and PID2020-112892RB-100, PID2020-113174-RA-100 [AEI/FEDER,UE], financed by MCIN/AEI/10.13039/501100011033), Cancer Research Institute under the CRI-CLIP, Asociación Española Contra el Cancer (AECC) Foundation under Grant GCB15152947MELE, Joint Translational Call for Proposals 2015 (JTC 2015) TRANSCAN-2 (code: TRS-2016-00000371), projects PI14/01686, PI13/00207, PI16/00668, PI19/01128, funded by Instituto de Salud Carlos III and co-funded by European Union (ERDF, “A way to make Europe”), European Commission within the Horizon 2020 Programme (PROCROP - 635122), Gobierno de Navarra Proyecto LINTERNA Ref: 0011–1411, Mark Foundation, Fundación BBVA and Fundación Olga Torres. AT is supported by the Ramon y Cajal program from the Spanish Ministry of Science (RYC2019-026406-I financiada por MCIN/AEI /10.13039/501100011033 y por El FSE invierte en tu futuro). Sí

Interleukin-8 (CXCL8) produced in the tumor microenvironment correlates with poor response to checkpoint inhibitors and is known to chemoattract and activate immunosuppressive myeloid leukocytes. In human cancer, IL8 mRNA levels correlate with IL1B and TNF transcripts. Both cytokines induced IL-8 functional expression from a broad variety of human cancer cell lines, primary colon carcinoma organoids, and fresh human tumor explants. Although IL8 is absent from the mouse genome, a similar murine axis in which TNFα and IL-1β upregulate CXCL1 and CXCL2 in tumor cells was revealed. Furthermore, intratumoral injection of TNFα and IL-1β induced IL-8 release from human malignant cells xenografted in immunodeficient mice. In all these cases, the clinically used TNFα blockers infliximab and etanercept or the IL-1β inhibitor anakinra was able to interfere with this pathogenic cytokine loop. Finally, in paired plasma samples of patients with cancer undergoing TNFα blockade with infliximab in a clinical trial, reductions of circulating IL-8 were substantiated.Significance:IL-8 attracts immunosuppressive protumor myeloid cells to the tumor microenvironment, and IL-8 levels correlate with poor response to checkpoint inhibitors. TNFα and IL-1β are identified as major inducers of IL-8 expression on malignant cells across cancer types and models in a manner that is druggable with clinically available neutralizing agents.This article is highlighted in the In This Issue feature, p. 2007

Supplementary Figure from A Therapeutically Actionable Protumoral Axis of Cytokines Involving IL-8, TNFα, and IL-1β

Supplementary figures 1-8 and supplementary table 1

IL12-based local gene therapy of cancer constitutes an active area of clinical research using plasmids, mRNAs, and viral vectors. To improve antitumor effects, we have experimentally tested the combination of mRNA constructs encoding IL12 and IL18. Moreover, we have used a form of IL18 [decoy-resistant IL18 (DR-18)] which has preserved bioactivity but does not bind to the IL18 binding protein decoy receptor. Both cytokines dramatically synergize to induce IFNγ release from mouse splenocytes, and, if systemically cotransferred to the liver, they mediate lethal toxicity. However, if given intratumorally to B16OVA tumor-bearing mice, the combination attains efficacy against the directly treated tumor and moderate tumor-delaying activity on distant noninjected lesions. Cotreatment was conducive to the presence of more activated CD8+ T cells in the treated and noninjected tumors. In keeping with these findings, the efficacy of treatment was contingent on the integrity of CD8+ T cells and cDC1 dendritic cells in the treated mice. Furthermore, efficacy of IL12 plus DR-18 local mRNA coinjection against distant concomitant tumors could be enhanced upon combination with anti–PD-1 mAb systemic treatment, thus defining a feasible synergistic immunotherapy strategy.

Interleukin-8 (CXCL8) produced in the tumor microenvironment correlates with poor response to checkpoint inhibitors and is known to chemoattract and activate immunosuppressive myeloid leukocytes. In human cancer, IL8 mRNA levels correlate with IL1B and TNF transcripts. Both cytokines induced IL-8 functional expression from a broad variety of human cancer cell lines, primary colon carcinoma organoids, and fresh human tumor explants. Although IL8 is absent from the mouse genome, a similar murine axis in which TNFα and IL-1β upregulate CXCL1 and CXCL2 in tumor cells was revealed. Furthermore, intratumoral injection of TNFα and IL-1β induced IL-8 release from human malignant cells xenografted in immunodeficient mice. In all these cases, the clinically used TNFα blockers infliximab and etanercept or the IL-1β inhibitor anakinra was able to interfere with this pathogenic cytokine loop. Finally, in paired plasma samples of patients with cancer undergoing TNFα blockade with infliximab in a clinical trial, reductions of circulating IL-8 were substantiated.Significance:IL-8 attracts immunosuppressive protumor myeloid cells to the tumor microenvironment, and IL-8 levels correlate with poor response to checkpoint inhibitors. TNFα and IL-1β are identified as major inducers of IL-8 expression on malignant cells across cancer types and models in a manner that is druggable with clinically available neutralizing agents.This article is highlighted in the In This Issue feature, p. 2007

Supplementary figures 1-5 Supplementary methods

The ability of conventional type-1 dendritic cells (cDC1) to cross-present tumor antigens to CD8+ T cells is critical for the induction of antitumor CTLs. Mice that are constitutively deficient in cDC1 cells have been reported to fail to respond to immunotherapy strategies based on checkpoint inhibitors. However, further work is needed to clarify the precise time during immunotherapy treatment that cDC1 cells are required for the beneficial effect of treatment. Here, we used a refined XCR1-DTR-Venus transgenic mouse model to acutely deplete cDC1 cells and trace their behavior using intravital microscopy. Diphtheria toxin–mediated cDC1 depletion prior to immunotherapy treatment with anti–PD-1 and/or anti-CD137 immunostimulatory mAbs completely ablated antitumor efficacy. The efficacy of adoptive T-cell therapy was also hampered by prior cDC1 depletion. After the onset of immunotherapy treatment, depletion of cDC1s only moderately reduced the therapeutic efficacy of anti–PD-1 and anti-CD137 mAbs. Intravital microscopy of liver-engrafted tumors revealed changes in the intratumoral behavior of cDC1 cells in mice receiving immunotherapy, and treatment with diphtheria toxin to deplete cDC1s impaired tumor T-cell infiltration and function. These results reveal that the functional integrity of the cDC1 compartment is required at the onset of various immunotherapies to successfully treat established tumors.Significance:These findings reveal the intratumoral behavior of cDC1 dendritic cells in transgenic mouse models and demonstrate that the efficacy of immunotherapy regimens is precluded by elimination of these cells.

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Interleukin-12-based local gene therapy of cancer constitutes an active area of clinical research using plasmids, mRNAs and viral vectors. To improve antitumor effects, we have experimentally tested the combination of mRNA constructs encoding interleukin-12 (IL-12) and interleukin-18 (IL-18). Moreover, we have used a form of IL-18 (DR-18) which has preserved bioactivity but does not bind to the IL-18BP decoy receptor. Both cytokines dramatically synergize to induce IFNγ release from mouse splenocytes, and, if systemically co-transferred to the liver, they mediate lethal toxicity. However, if given intratumorally to B16OVA tumor-bearing mice, the combination attains efficacy against the directly treated tumor and moderate tumor-delaying activity on distant uninjected lesions. Co-treatment was conducive to the presence of more activated CD8+ T cells in the treated and non-injected tumors. In keeping with these findings, the efficacy of treatment was contingent on the integrity of CD8+ T cells and cDC1 dendritic cells in the treated mice. Furthermore, efficacy of IL-12 plus DR-18 local mRNA co-injection against distant concomitant tumors could be enhanced upon combination with anti-PD-1 mAb systemic treatment, thus defining a feasible synergistic immunotherapy strategy.

Cytokines are pleiotropic soluble proteins used by immune cells to orchestrate a coordinated response against pathogens and malignancies. In cancer immunotherapy, cytokine-based drugs can be developed potentiating pro-inflammatory cytokines or blocking immunosuppressive cytokines. However, the complexity of the mechanisms of action of cytokines requires the use of biotechnological strategies to minimize systemic toxicity, while potentiating the antitumor response. Sequence mutagenesis, fusion proteins and gene therapy strategies are employed to enhance the half-life in circulation, target the desired bioactivity to the tumor microenvironment, and to optimize the therapeutic window of cytokines. In this review, we provide an overview of the different strategies currently being pursued in pre-clinical and clinical studies to make the most of cytokines for cancer immunotherapy.

BackgroundOn the basis of efficacy in mouse tumor models, multiple CD137 (4-1BB) agonist agents are being preclinically and clinically developed. The costimulatory molecule CD137 is inducibly expressed as a transmembrane or as a soluble protein (sCD137). Moreover, the CD137 cytoplasmic signaling domain is a key part in approved chimeric antigen receptors (CARs). Reliable pharmacodynamic biomarkers for CD137 ligation and costimulation of T cells will facilitate clinical development of CD137 agonists in the clinic.MethodsWe used human and mouse CD8 T cells undergoing activation to measure CD137 transcription and protein expression levels determining both the membrane-bound and soluble forms. In tumor-bearing mice plasma sCD137 concentrations were monitored on treatment with agonist anti-CD137 monoclonal antibodies (mAbs). Human CD137 knock-in mice were treated with clinical-grade agonist anti-human CD137 mAb (Urelumab). Sequential plasma samples were collected from the first patients intratumorally treated with Urelumab in the INTRUST clinical trial. Anti-mesothelin CD137-encompassing CAR-transduced T cells were stimulated with mesothelin coated microbeads. sCD137 was measured by sandwich ELISA and Luminex. Flow cytometry was used to monitor CD137 surface expression.ResultsCD137 costimulation upregulates transcription and protein expression of CD137 itself including sCD137 in human and mouse CD8 T cells. Immunotherapy with anti-CD137 agonist mAb resulted in increased plasma sCD137 in mice bearing syngeneic tumors. sCD137 induction is also observed in human CD137 knock-in mice treated with Urelumab and in mice transiently humanized with T cells undergoing CD137 costimulation inside subcutaneously implanted Matrigel plugs. The CD137 signaling domain-containing CAR T cells readily released sCD137 and acquired CD137 surface expression on antigen recognition. Patients treated intratumorally with low dose Urelumab showed increased plasma concentrations of sCD137.ConclusionsCD137 in plasma and CD137 surface expression can be used as quantitative parameters dynamically reflecting therapeutic costimulatory activity elicited by agonist CD137-targeted agents.

Introduction: Immune checkpoint inhibitors and adoptive T-cell therapy based on chimeric antigen receptors are the spearhead strategies to exploit the immune system to fight cancer. To take advantage of the full potential of the immune system, cancer immunotherapy must incorporate new biotechnologies such as mRNA technology that may synergize with already approved immunotherapies and act more effectively on immune targets. Areas covered: This review describes the basics of mRNA biotechnology and provides insight into the recent advances in the use of mRNA for the local and systemic delivery of immunostimulatory antibodies, proinflammatory cytokines or for optimizing adoptive T-cell therapy.Expert opinion: mRNA-based nanomedicines have great potential to expand the arsenal of immunotherapy tools due to their ability to simplify and accelerate drug development and their suitability for transient and local expression of immunostimulatory molecules, whose systemic and sustained expression would be toxic. The success of mRNA-based COVID-19 vaccines has highlighted the feasibility of this approach. Continuous advances in the delivery and construction of RNA-based vectors hold promise for improvements in clinical efficacy.

Interleukin-12 is considered a potent agent to enhance antitumor immune responses. It belongs to a family of heterodimeric cytokines with key roles in the up-regulation and down-regulation of cellular immunity. Since its discovery, recombinant IL-12 was found to exert potent antitumor effects in rodent tumor models and was rapidly tested in the clinic with an unfavorable benefit/toxicity profile. Localized delivery of IL-12 dramatically improves the therapeutic index and this approach is being applied in the clinic based on in-vivo electroporation of naked plasmid DNA encoding IL-12, mRNA formulations, viral vectors and tumor-targeted fusion proteins. Other biotechnology strategies such as IL-12-engineered local adoptive cell therapy and pro-cytokines can also be used to improve results and broaden the therapeutic window. Combination strategies of such localized IL-12-based approaches with checkpoint inhibitors are yielding promising results both preclinically and in the early-phase clinical trials.

IL12 is a very potent cancer immunotherapy agent, but is difficult to harness safely if given systemically. Local gene transfer aims to confine the effects of IL12 to malignant tissues, thus avoiding toxicity. Lipid-nanoparticle mRNA achieves IL12 expression and efficacy in mouse models, opening the way to an ongoing trial. See related article by Hewitt et al., p. 6284

Costimulation via CD137 (4-1BB) enhances antitumor immunity mediated by cytotoxic T lymphocytes. Anti-CD137 agonist antibodies elicit mild liver inflammation in mice, and the maximum tolerated dose of Urelumab, an anti-human CD137 agonist monoclonal antibody, in the clinic was defined by liver inflammation–related side effects. A protease-activated prodrug form of the anti-mouse CD137 agonist antibody 1D8 (1D8 Probody therapeutic, Pb-Tx) was constructed and found to be selectively activated in the tumor microenvironment. This construct, which encompasses a protease-cleavable linker holding in place a peptide that masks the antigen binding site, exerted antitumor effects comparable to the unmodified antibody but did not result in liver inflammation. Moreover, it efficaciously synergized with both PD-1 blockade and adoptive T-cell therapy. Surprisingly, minimal active Pb-Tx reached tumor-draining lymph nodes, and regional lymphadenectomy did not abrogate antitumor efficacy. By contrast, S1P receptor–dependent recirculation of T cells was absolutely required for efficacy. The preferential cleavage of the anti-CD137 Pb-Tx by tumor proteases offers multiple therapeutic opportunities, including neoadjuvant therapy, as shown by experiments in which the Pb-Tx is given prior to surgery to avoid spontaneous metastases.

Simple Summary Peritoneal carcinomatosis mouse models as a platform to test, improve and/or predict the appropriate therapeutic interventions in patients are crucial to providing medical advances. Here, we overview reported mouse models to explore peritoneal carcinomatosis in translational biomedical research. Abstract Peritoneal carcinomatosis of primary tumors originating in gastrointestinal (e.g., colorectal cancer, gastric cancer) or gynecologic (e.g., ovarian cancer) malignancies is a widespread type of tumor dissemination in the peritoneal cavity for which few therapeutic options are available. Therefore, reliable preclinical models are crucial for research and development of efficacious treatments for this condition. To date, a number of animal models have attempted to reproduce as accurately as possible the complexity of the tumor microenvironment of human peritoneal carcinomatosis. These include: Syngeneic tumor cell lines, human xenografts, patient-derived xenografts, genetically induced tumors, and 3D scaffold biomimetics. Each experimental model has its own strengths and limitations, all of which can influence the subsequent translational results concerning anticancer and immunomodulatory drugs under exploration. This review highlights the current status of peritoneal carcinomatosis mouse models for preclinical development of anticancer drugs or immunotherapeutic agents.

In humans, IL-8 (CXCL8) is a key chemokine for chemotaxis of polymorphonuclear leukocytes and monocytes/macrophages when acting on CXCR1 and CXCR2. CXCL8 activity on neutrophils includes chemotaxis and eliciting the extrusion of neutrophil extracellular traps (NETs). In this study, we show that concentrations of IL-8 that induce NETosis surpass in at least one order of magnitude those required to elicit chemoattraction in human neutrophils. IL-8-induced NETosis was less dependent on G-proteins than migration, while extracellular Ca+2 chelation similarly inhibited both processes. Reactive oxygen species (ROS) were more important for NETosis than for chemotaxis as evidenced by neutralization with N-acetyl -cysteine. Interestingly, selective blockade with anti-CXCR1 mAb inhibited NETosis much more readily than chemotaxis, while pharmacological inhibition of both CXCR1 and CXCR2, or selective inhibition for CXCR2 alone, similarly inhibited both functions. Together, these results propose a model according to which low concentrations of IL-8 in a gradient attract neutrophils to the inflammatory foci, while high receptor-saturating concentrations of IL-8 give rise to NETosis once leukocytes reach the core of the inflammatory insult.

Summary Objectives: Cachexia is a systemic metabolic disorder characterized by loss of fat and muscle mass, which disproportionately impacts patients with gastrointestinal malignancies such as pancreatic cancer. While the immunologic shifts contributing to the development of other adipose tissue (AT) pathologies such as obesity have been well described, the immune microenvironment has not been studied in the context of cachexia. Methods: We performed bulk RNA-sequencing, cytokine arrays, and flow cytometry to characterize the immune landscape of visceral AT (VAT) in the setting of pancreatic and colorectal cancers. Results: The cachexia inducing factor IL-6 is strongly elevated in the wasting VAT of cancer bearing mice, but the regulatory type 2 immune landscape which characterizes healthy VAT is maintained. Pathologic skewing toward Th1 and Th17 inflammation is absent. Similarly, the VAT of patients with colorectal cancer is characterized by a Th2 signature with abundant IL-33 and eotaxin-2, albeit also with high levels of IL-6. Conclusions: Wasting AT during the development of cachexia may not undergo drastic changes in immune composition like those seen in obese AT. Our approach provides a framework for future immunologic analyses of cancer associated cachexia., Graphical Abstract Graphical Abstract

One of the most important mechanisms by which cancer fosters its own development is the generation of an immune microenvironment that inhibits or impairs antitumor immune responses. A cancer permissive immune microenvironment is present in a large proportion of the patients with cancer who do not respond to immunotherapy approaches intended to trigger preexisting antitumor immune responses, for instance, immune checkpoint blockade. High circulating levels of IL8 in patients with cancer quite accurately predict those who will not benefit from checkpoint-based immunotherapy. IL8 has been reported to favor cancer progression and metastases via different mechanisms, including proangiogenesis and the maintenance of cancer stem cells, but its ability to attract and functionally modulate neutrophils and macrophages is arguably one of the most important factors. IL8 does not only recruit neutrophils to tumor lesions, but also triggers the extrusion of neutrophil extracellular traps (NET). The relevance and mechanisms underlying the contribution of both neutrophils and NETs to cancer development and progression are starting to be uncovered and include both direct effects on cancer cells and changes in the tumor microenvironment, such as facilitating metastasis, awakening micrometastases from dormancy, and facilitating escape from cytotoxic immune cells. Blockade of IL8 or its receptors (CXCR1 and CXCR2) is being pursued in drug development, and clinical trials alone or in combination with anti-PD-L1 checkpoint inhibitors are already ongoing.

Background: Interleukin-8 produced in the tumor microenvironment correlates with poor response to checkpoint inhibitors and is known to chemoattract and activate immunosuppressive myeloid leukocytes. An important question is what are the mechanisms that control IL-8 transcription in tumors. Various factors have been described but the main common denominator is NF-кB activation. In this regard, considering inflammatory mediators present in the tumor and whose receptors act as NF-кB inducers, TNFα and IL-1β became obvious candidates. In this study, we provide multiple lines of evidence that TNFα and IL-1β are able to elicit functional IL-8 from malignant cells in a manner that can be inhibited by clinically grade blockade agents. Methods: Short-term cultured human tumor cell lines, fresh human tumor explants and primary organoids were stimulated with TNFα or IL-1β to test for IL-8 induction. Moreover, syngeneic and xenografted tumors were also used to study IL-8 induction in response to TNFα or to IL-1β. Correlation between these cytokines and IL-8 transcripts were studied in TCGA and other databases to search. Results: We have been able to correlate IL-8 mRNA levels with IL-1β and TNFα transcripts in human cancers. Both cytokines induced IL-8 functional expression from a broad variety of human cancer cell lines, primary colon carcinoma organoids and fresh human tumor explants. Although IL-8 is absent from the mouse genome, a similar murine axis in which TNFα and IL-1β upregulate CXCL1 and CXCL2 in several mouse tumor cells was revealed. Furthermore, intratumoral injection of TNFα and IL-1β induced IL-8 release from human malignant cells xenografted in immunodeficient mice. In all these cases, the clinically used TNFα-blockers infliximab and etanercept, or the IL-1β inhibitor anakinra were able to interfere with this pathogenic cytokine loop. Moreover, treatment with checkpoint inhibitors augments TNFα in syngeneic and xenografted tumors thereby turning on this immunosuppressive chemokine-driven axis. However, we have demonstrated that co-blockade of TNFα and IL-1β could equally enhance anti-tumor efficacy of αPD1+αCTLA-4 regimens as CXCR1/CXCR2 inhibitors, reparixin and AZD5069, did. Conclusions: In this study, we have identified TNFα and IL-1β as major inducers of IL-8 transcription on human cancers. In keeping with these findings, both TNFα and IL-1β induce functional IL-8 release from all tested human tumor cell lines representing a variety of tissue origins, as well as from primary organoids and fresh human cancer explants. In all these cases, including xenografted human tumors, IL-8 induction could be blunted by the pharmacological blockade of TNFα or IL-1β. Finally, inhibition of this pathogenic cytokine loop could increase the anti-tumoral effect of checkpoint inhibitors. Citation Format: Irene Olivera, Rebeca Sanz-Pamplona, Elixabet Bolaños, Inmaculada Rodriguez, Iñaki Etxeberria, Assunta Cirella, Saray Garasa, Itziar Migueliz, Iñaki Eguren, Miguel F. Sanmamed, Javier Glez-Vaz, Arantza Azpilikueta, Maite Alvarez, Maria C. Ochoa, Carlos E. De Andrea, Pedro Berraondo, Álvaro Teijeira, Ignacio Melero. A therapeutically actionable pro-tumoral axis of cytokines involving interleukin-8, TNFα and IL-1β [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3826.

Background: On the basis of efficacy in mouse tumor models, multiple CD137 (4-1BB) agonist agents are being preclinically and clinically developed. The costimulatory molecule CD137 is inducibly expressed as a transmembrane or as a soluble protein (sCD137). Moreover, the CD137 cytoplasmic signaling domain is a key part in approved Chimeric Antigen Receptors (CARs). Reliable pharmacodynamic biomarkers for CD137 ligation and co-stimulation of T cells will facilitate clinical development of CD137 agonists in the clinic. Methods: We used human and mouse CD8 T cells undergoing activation to measure CD137 transcription and protein expression levels determining both of the membrane-bound and soluble forms. In tumor-bearing mice plasma sCD137 concentrations were monitored upon treatment with agonist anti-CD137 mAbs. Human CD137 knock-in mice were treated with clinical-grade agonist anti-human CD137 (Urelumab). Sequential plasma samples were collected from the first patients intratumorally treated with Urelumab in the INTRUST clinical trial. Anti-Mesothelin CD137-encompassing CAR-transduced T cells were stimulated with mesothelin coated microbeads. sCD137 was measured by sandwich ELISAs and Luminex and flow cytometry was used to monitor CD137 surface expression. Results: CD137 co-stimulation upregulates transcription and protein expression of CD137 itself including sCD137 in human and mouse CD8 T cells. Immunotherapy with anti-CD137 agonist mAb resulted in increased plasma sCD137 in mice bearing syngeneic tumors. sCD137 induction is also observed in human CD137 knock-in mice treated with Urelumab and in mice transiently humanized with T cells undergoing CD137 co-stimulation inside subcutaneous implanted Matrigel® plugs. CD137 signaling domain-containing CAR T cells readily released sCD137 upon antigen recognition in a manner favored by the CD137 signaling domain. Patients treated intratumorally with low dose Urelumab showed increased plasma concentrations of sCD137. Conclusion: sCD137 in plasma and CD137 surface expression can be used as quantitative parameters dynamically reflecting therapeutic co-stimulatory activity elicited by agonist CD137-targeted agents. Citation Format: Javier Glez-Vaz, Arantza Azpilikueta, Irene Olivera, Assunta Cirella, Álvaro Teijeira, Carmen Ochoa, Maite Álvarez, Iñaki Eguren, Carlos Luri-Rey, Miguel F. Sanmamed, Ignacio Melero. Soluble CD137 as a dynamic biomarker to monitor agonist CD137 immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 628.

Introduction Nutrition and physical activity are key elements in the prevention and management of type 2 diabetes. A community-based, multidisciplinary educational intervention aimed to improve quality of life and disease self-management in sedentary, overweight/obese type 2 diabetic patients was implemented in Naples, South Italy. Methods The 9-month intervention included a motivational program, a nutrition program, and an exercise program. Satisfaction, worry, and embarrassment regarding their condition, together with disease-related behaviors and propensity towards physical activity, were evaluated through a validated questionnaire before and after the intervention; health status perception was evaluated through the short-form 12 questionnaire. Changes in HbA1c level and weight were also checked. Results A significant improvement (p

IntroductionUnhealthy lifestyles may hinder bariatric surgery outcomes. This non-randomized controlled study aimed to evaluate the effects of an integrated post-operative exercise-based educational and motivational program in improving behaviors, quality of life, anthropometric features, cardiorespiratory and physical fitness in bariatric patients respect to the only surgical intervention.MethodsA group of adult sedentary bariatric patients chose to attend a 12-month exercise program integrated with diet education and motivational support, or to receive usual care. Dietary habits, binge eating disorder, physical activity, obesity-related quality of life, Body Mass Index, waist and hip circumference, VO2max, strength and flexibility were assessed at the start and at the end of the study in both groups.ResultsOn a total of 82 patients enrolled, follow-up measures were obtained from 28 (85.7% females, mean age 38.2±8.7) and 42 (71.4% females, mean age 40.2±9.5) patients included in the intervention and control group, respectively. All the behavioral and physical outcomes improved significantly in the participants to the intervention, while the control group showed lesser changes, especially regarding quality of life and physical fitness.ConclusionsNotwithstanding the self-selection, these results suggest that a timely postoperative behavioral multidisciplinary program for bariatric patients may be effective in establishing healthy behaviors which can lead to better surgery outcomes.

Breast cancer is the most frequent malignancy in females in terms of both incidence and mortality. Underlying the high mortality rate is the presence of cancer stem cells, which divide indefinitely and are resistant to conventional chemotherapies, so causing tumor relapse. In the present study, we identify miR-216a-5p as a downregulated microRNA in breast cancer stem cells vs. the differentiated counterpart. We demonstrate that overexpression of miR-216a-5p impairs stemness markers, mammosphere formation, ALDH activity, and the level of Toll-like receptor 4 (TLR4), which plays a significant role in breast cancer progression and metastasis by leading to the release of pro-inflammatory molecules, such as interleukin 6 (IL-6). Indeed, miR-216a regulates the crosstalk between cancer cells and the cells of the microenvironment, in particular cancer-associated fibroblasts (CAFs), through regulation of the TLR4/IL6 pathway. Thus, miR-216a has an important role in the regulation of stem phenotype, decreasing stem-like properties and affecting the cross-talk between cancer cells and the tumor microenvironment.

Background One limitation in the breast cancer research field is that there are few in vitro models of breast cancer able to predict drug response and thus clinical patients’ outcome, mainly because the present models do not take account of the tumor heterogeneity. To address this issue, we developed an in vitro 3D organoid culture system using primary human breast cancer tissue that could be used to recapitulate in vivo organs. A major difficulty in the development of such models is to identify in vitro conditions that preserve the breast cancer phenotypes observed in vivo. Methods We isolated organoids from breast cancer patients and optimized the organoids growing conditions. By immunofluorescence assay we confirmed the receptor status, and the preservation of epithelial (E-cadherin) or the fibroblasts components included in the tissue microenvironment (TME) (alfa-SMA and FAP). Here we used organoids as mimic of tumor burden for drug penetration studies. In particular, we studied doxorubicin (DOX) penetration and biodistribution, by several means: immunofluorescence studies, caspase assay, and cell viability. Results We demonstrated by IF studies, that miR-340, an oncosuppressor miR, was able to modulate DOX penetration. At the same time, miR-340 induced an increase in DOX sensitivity assessed by caspase-3 assay, PARP cleavage, as well as p38 phosphorylation. Breast cancer organoids have also been used to better understand the role of the TME in breast cancer response to therapy. We assessed drug sensitivity in the presence or absence of conditioned media obtained from cancer activated fibroblasts (CAFs). We found that organoids treated with conditioned media exhibit lower level of caspase 3 activation compared to the control upon palbociclib treatment, suggesting the importance role of TME in drug resistance. Conclusions Thus, organoids can be considered as a new tool for studying breast cancer and developing personalized medicine approaches and to test possible use of RNA therapeutics. Legal entity responsible for the study Gerolama Condorelli. Funding AIRC. Disclosure All authors have declared no conflicts of interest.

Background and Aims: Personality disorders are frequently associated with eating disorders in obese patients and may negatively affect weight loss and maintenance after bariatric surgery. This non-randomized study aimed to assess the effects of different psychotherapeutic interventions on weight loss in a sample of patients with borderline personality disorder who underwent laparoscopic gastric bypass or laparoscopic adjustable gastric banding. Materials and Methods: A total of 153 bariatric patients meeting borderline personality disorder criteria were chosen voluntarily and consecutively to undergo an interpersonal individual treatment (n = 50), a dialectical behavioral group treatment (n = 50), or treatment as usual (n = 53) for a year after surgery. Their body mass index was measured before and at the end of each treatment. Results and Conclusion: A total of 12 patients (7.8%) dropped out of the study. Significantly higher body mass index reductions were registered in both experimental groups (−14.2 and −9.4 kg/m2, respectively) compared with the treatment as usual group (−2.1 kg/m2; p < 0.01). Treated patients who underwent laparoscopic gastric bypass (total n = 94) showed better outcomes than those who underwent laparoscopic adjustable gastric banding (total n = 45), while no differences were observed in untreated patients. This study demonstrates the role of pre-operative psychological assessment and post-operative psychotherapeutic support in improving weight loss among bariatric patients with borderline personality disorder. A randomized controlled trial is needed to confirm these preliminary findings.

Borderline personality disorder (BPD) and eating disorders may affect the outcomes of bariatric surgery. This study was aimed to evaluate the influence of a post-operative dialectical behavioral therapy (DBT) intervention on weight loss and comorbidities in a sample of comorbid obese Italian patients with BPD traits and bulimia tendencies who underwent laparoscopic Roux-en-Y gastric bypass (LRYGB) and laparoscopic adjustable gastric banding (LAGB). One hundred fifty-four LRYGB or LAGB patients showing BPD and bulimia traits and type II diabetes, hypertension, and/or sleep apnea (OSAS) voluntarily adhered to DBT (n = 72) or treatment as usual (n = 82) for 12 months after surgery. Total weight loss (%TWL) and remission/improvement of comorbidities were considered as outcomes. Differences between treatment groups and between LRYGB and LAGB subgroups were evaluated through Student’s t test for TWL and chi-square test for comorbidities. DBT was more effective than treatment as usual (TAU) in reducing weight loss (p

Breast cancer is one of the leading causes of cancer mortality among women. The main causes of death are related to recurrence and metastasis. One major limitation in the breast cancer research field is that there are few in vitro models of breast cancer able to predict clinical patients' outcome. The main reason of this failure is that the present models do not take complexity or heterogeneity into account. Therefore, innovative models are now required to better represent tumor fidelity. To address this issue, we developed an in vitro 3D organoid culture system using primary human breast cancer tissue. Organoids are miniature forms of tissues that exhibit three-dimensional architecture and are able to maintain phenotypical properties cultured in a dish. Therefore, they could be used as models for diseases, including cancer. A major difficulty in the development of such models is to identify in vitro conditions that preserve the breast cancer phenotypes observed in vivo. For this reason, we isolated organoids from breast cancer patients and optimized the organoids growing conditions. We used a specialised medium and matrix (Cultrex Trevigen) required for supporting cell growth as it occurs in the human body. Then, we decided to test whether the obtained organoids could be used as an in vitro prediction tool. First, we performed immunofluorescence assay to assess the preservation of patient architecture (CK14, CK18 and E cadherin) and clinical phenotype (HER2, ER, PR). Then, we used an innovative chemotaxis assay in 3D collagen gels for analysing organoids cell invasion. It is based on a chamber in which a chemoattractant gradient is generated by diffusion through a porous membrane for mimicking blood microenvironment. Then we analysed the morphological changes after chemotherapeutics treatment to correlate the organoids response with patient tumor behaviour. Thus, organoids can be considered as a new tool for studying breast cancer and developing personalized medicine approaches for this disease. Citation Format: Giuseppina Roscigno, Assunta Cirella, Alessandra Affinito, Francesco Palma, Maurizio Albero, Martina Messere, Martina Gelormini, Lorenza Cocca, Renato Thomas, Gerolama Condorelli. Organoids: A new tool for the prediction of breast cancer patients' outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1170A.