Harnessing high density lipoproteins to block transforming growth factor beta and to inhibit the growth of liver tumor metastases

Transforming growth factor β (TGF-β) is a powerful promoter of cancer progression and a key target for antitumor therapy. As cancer cells exhibit active cholesterol metabolism, high density lipoproteins (HDLs) appear as an attractive delivery system for anticancer TGFβ-inhibitory molecules. We constructed a plasmid encoding a potent TGF-β-blocking peptide (P144) linked to apolipoprotein A-I (ApoA-I) through a flexible linker (pApoLinkerP144). The ApoLinkerP144 sequence was then incorporated into a hepatotropic adeno-associated vector (AAVApoLinkerP144). The aim was to induce hepatocytes to produce HDLs containing a modified ApoA-I capable of blocking TGF-β. We observed that transduction of the murine liver with pApoLinkerP144 led to the appearance of a fraction of circulating HDL containing the fusion protein. These HDLs were able to attenuate TGF-β signaling in the liver and to enhance IL-12 -mediated IFN-γ production. Treatment of liver metastasis of MC38 colorectal cancer with AAVApoLinkerP144 resulted in a significant reduction of tumor growth and enhanced expression of IFN-γ and GM-CSF in cancerous tissue. ApoLinkerP144 also delayed MC38 liver metastasis in Rag2-/-IL2rγ-/- immunodeficient mice. This effect was associated with downregulation of TGF-β target genes essential for metastatic niche conditioning. Finally, in a subset of ret transgenic mice, a model of aggressive spontaneous metastatic melanoma, AAVApoLinkerP144 delayed tumor growth in association with increased CD8+ T cell numbers in regional lymph nodes. In conclusion, modification of HDLs to transport TGF-β-blocking molecules is a novel and promising approach to inhibit the growth of liver metastases by immunological and non-immunological mechanisms

Kinetic and dynamic computational model-based characterization of new proteins in mice: application to interferon alpha linked to apolipoprotein a-I

Interferon alpha linked to apolipoprotein A-I has been recently proposed as an improved interferon-based therapy. In the present study, we aimed to develop a computational model to gain further insight into the in vivo behaviour of this new fusion protein. In order to facilitate in vivo evaluation of interferon and the fusion protein without altering their biological properties, green fluorescent protein was incorporated into their structures. Kinetic and dynamic behaviour of both compounds was successfully described after plasmid hydrodynamic administration and in situ synthesis of the studied proteins. Results from the modelling exercise showed that apolipoprotein A-I conferred a modified kinetic behaviour, varying molecule distribution and prolonging half-life without altering liver dynamic performance. However, differences in the gene expression activity were observed at brain level between both compounds. Those differences could be explained by modifications in the dynamic, but also in the biodistribution properties, which would be worth evaluating in future experiments. Therefore, the modelling approach provided a global comprehension of a complex system and allowed us to compare the in vivo behaviour of both compounds and to identify critical aspects that might be important to understand the system better and suggests a need for new model-based experiments

Harnessing high density lipoproteins to block transforming growth factor beta and to inhibit the growth of liver tumor metastases

Transforming growth factor β (TGF-β) is a powerful promoter of cancer progression and a key target for antitumor therapy. As cancer cells exhibit active cholesterol metabolism, high density lipoproteins (HDLs) appear as an attractive delivery system for anticancer TGFβ-inhibitory molecules. We constructed a plasmid encoding a potent TGF-β-blocking peptide (P144) linked to apolipoprotein A-I (ApoA-I) through a flexible linker (pApoLinkerP144). The ApoLinkerP144 sequence was then incorporated into a hepatotropic adeno-associated vector (AAVApoLinkerP144). The aim was to induce hepatocytes to produce HDLs containing a modified ApoA-I capable of blocking TGF-β. We observed that transduction of the murine liver with pApoLinkerP144 led to the appearance of a fraction of circulating HDL containing the fusion protein. These HDLs were able to attenuate TGF-β signaling in the liver and to enhance IL-12 -mediated IFN-γ production. Treatment of liver metastasis of MC38 colorectal cancer with AAVApoLinkerP144 resulted in a significant reduction of tumor growth and enhanced expression of IFN-γ and GM-CSF in cancerous tissue. ApoLinkerP144 also delayed MC38 liver metastasis in Rag2-/-IL2rγ-/- immunodeficient mice. This effect was associated with downregulation of TGF-β target genes essential for metastatic niche conditioning. Finally, in a subset of ret transgenic mice, a model of aggressive spontaneous metastatic melanoma, AAVApoLinkerP144 delayed tumor growth in association with increased CD8+ T cell numbers in regional lymph nodes. In conclusion, modification of HDLs to transport TGF-β-blocking molecules is a novel and promising approach to inhibit the growth of liver metastases by immunological and non-immunological mechanisms

Eradication of large tumors expressing human papillomavirus E7 protein by therapeutic vaccination with E7 fused to the extra domain a from fibronectin

Cervical carcinoma is one of the most common cancers in women worldwide. It is well established that chronic infection of the genital tract by various mucosatropic human papillomavirus (HPV) types causes cervical cancer. Cellular immunity to E7 protein from HPV (HPVE7) has been associated with clinical and cytologic resolution of HPV-induced lesions. Thus, we decided to test if targeting of HPVE7 to dendritic cells using a fusion protein containing the extra domain A (EDA) from fibronectin, a natural ligand for TLR4, and HPVE7 (EDA-HPVE7) might be an efficient vaccine for the treatment of cervical carcinoma. We found that EDA-HPVE7 fusion protein was efficiently captured by bone marrow derived dendritic cells in vitro and induced their maturation, with the upregulation of maturation markers and the production of IL-12. Immunization of mice with EDA-HPVE7 fusion protein induced antitumor CD8(+) T cell responses in the absence of additional adjuvants. Repeated intratumoral administration of EDA-HPVE7 in saline was able to cure established TC-1 tumors of 5-7 mm in diameter. More importantly, intravenous injection with EDA-HPVE7 in combination with the TLR ligand polyinosinic-polycytidylic acid (pIC), or with low doses of cyclophosphamide and the TLR9 ligand CpG-B complexed in cationic lipids, were able to eradicate large established TC-1 tumors (1.2 cm in diameter). Thus, therapeutic vaccination with EDA-HPVE7 fusion protein may be effective in the treatment of human cervical carcinom

Lactate dehydrogenase inhibition synergizes with IL-21 to promote CD8+T cell stemness and antitumor immunity

Interleukin (IL)-2 and IL-21 dichotomously shape CD8(+) T cell differentiation. IL-2 drives terminal differentiation, generating cells that are poorly effective against tumors, whereas IL-21 promotes stem cell memory T cells (T-SCM) and antitumor responses. Here we investigated the role of metabolic programming in the developmental differences induced by these cytokines. IL-2 promoted effector-like metabolism and aerobic glycolysis, robustly inducing lactate dehydrogenase (LDH) and lactate production, whereas IL-21 maintained a metabolically quiescent state dependent on oxidative phosphorylation. LDH inhibition rewired IL-2-induced effects, promoting pyruvate entry into the tricarboxylic acid cycle and inhibiting terminal effector and exhaustion programs, including mRNA expression of members of the NR4A family of nuclear receptors, as well as Prdm1 and Xbp1. While deletion of Ldha prevented development of cells with antitumor effector function, transient LDH inhibition enhanced the generation of memory cells capable of triggering robust antitumor responses after adoptive transfer. LDH inhibition did not significantly affect IL-21-induced metabolism but caused major transcriptomic changes, including the suppression of IL-21-induced exhaustion markers LAG3, PD1, 2B4, and TIM3. LDH inhibition combined with IL-21 increased the formation of TSCM cells, resulting in more profound antitumor responses and prolonged host survival. These findings indicate a pivotal role for LDH in modulating cytokine-mediated T cell differentiation and underscore the therapeutic potential of transiently inhibiting LDH during adoptive T cell-based immunotherapy, with an unanticipated cooperative antitumor effect of LDH inhibition and IL-21

Stoma-free Survival After Rectal Cancer Resection With Anastomotic Leakage: Development and Validation of a Prediction Model in a Large International Cohort.

Objective:To develop and validate a prediction model (STOMA score) for 1-year stoma-free survival in patients with rectal cancer (RC) with anastomotic leakage (AL).Background:AL after RC resection often results in a permanent stoma.Methods:This international retrospective cohort study (TENTACLE-Rectum) encompassed 216 participating centres and included patients who developed AL after RC surgery between 2014 and 2018. Clinically relevant predictors for 1-year stoma-free survival were included in uni and multivariable logistic regression models. The STOMA score was developed and internally validated in a cohort of patients operated between 2014 and 2017, with subsequent temporal validation in a 2018 cohort. The discriminative power and calibration of the models' performance were evaluated.Results:This study included 2499 patients with AL, 1954 in the development cohort and 545 in the validation cohort. Baseline characteristics were comparable. One-year stoma-free survival was 45.0% in the development cohort and 43.7% in the validation cohort. The following predictors were included in the STOMA score: sex, age, American Society of Anestesiologist classification, body mass index, clinical M-disease, neoadjuvant therapy, abdominal and transanal approach, primary defunctioning stoma, multivisceral resection, clinical setting in which AL was diagnosed, postoperative day of AL diagnosis, abdominal contamination, anastomotic defect circumference, bowel wall ischemia, anastomotic fistula, retraction, and reactivation leakage. The STOMA score showed good discrimination and calibration (c-index: 0.71, 95% CI: 0.66-0.76).Conclusions:The STOMA score consists of 18 clinically relevant factors and estimates the individual risk for 1-year stoma-free survival in patients with AL after RC surgery, which may improve patient counseling and give guidance when analyzing the efficacy of different treatment strategies in future studies

Stoma-free survival after anastomotic leak following rectal cancer resection: worldwide cohort of 2470 patients

Background: The optimal treatment of anastomotic leak after rectal cancer resection is unclear. This worldwide cohort study aimed to provide an overview of four treatment strategies applied. Methods: Patients from 216 centres and 45 countries with anastomotic leak after rectal cancer resection between 2014 and 2018 were included. Treatment was categorized as salvage surgery, faecal diversion with passive or active (vacuum) drainage, and no primary/secondary faecal diversion. The primary outcome was 1-year stoma-free survival. In addition, passive and active drainage were compared using propensity score matching (2: 1). Results: Of 2470 evaluable patients, 388 (16.0 per cent) underwent salvage surgery, 1524 (62.0 per cent) passive drainage, 278 (11.0 per cent) active drainage, and 280 (11.0 per cent) had no faecal diversion. One-year stoma-free survival rates were 13.7, 48.3, 48.2, and 65.4 per cent respectively. Propensity score matching resulted in 556 patients with passive and 278 with active drainage. There was no statistically significant difference between these groups in 1-year stoma-free survival (OR 0.95, 95 per cent c.i. 0.66 to 1.33), with a risk difference of -1.1 (95 per cent c.i. -9.0 to 7.0) per cent. After active drainage, more patients required secondary salvage surgery (OR 2.32, 1.49 to 3.59), prolonged hospital admission (an additional 6 (95 per cent c.i. 2 to 10) days), and ICU admission (OR 1.41, 1.02 to 1.94). Mean duration of leak healing did not differ significantly (an additional 12 (-28 to 52) days). Conclusion: Primary salvage surgery or omission of faecal diversion likely correspond to the most severe and least severe leaks respectively. In patients with diverted leaks, stoma-free survival did not differ statistically between passive and active drainage, although the increased risk of secondary salvage surgery and ICU admission suggests residual confounding