Oxaliplatin in combination with liver-specific expression of interleukin 12 reduces the immunosuppressive microenvironment of tumours and eradicates metastatic colorectal cancer in mice

BACKGROUND AND AIMS: New options are needed for the management and prevention of colorectal cancer liver metastases. Interleukin 12 (IL-12) is an immunostimulatory cytokine with proven antitumour effect in animal models. Despite evidence indicating its biological effect in humans, neither the recombinant protein nor gene therapy vectors expressing IL-12 have shown a relevant benefit in patients with cancer. OBJECTIVE: To develop a new approach to overcome the difficulties in obtaining a suitable expression pattern and the immunosuppressive milieu in the tumours which contribute to this poor performance. METHODS: A high-capacity ('gutless') adenoviral vector carrying a liver-specific, mifepristone (Mif)-inducible system for the expression of IL-12 (HC-Ad/RUmIL-12) was used in combination with chemotherapy. Tumours were established in the liver of C57BL/6 mice by inoculation of MC38 colon cancer cells. RESULTS: Intrahepatic injection of HC-Ad/RUmIL-12 and tailored induction regimens allowed the maintenance of safe and efficient levels of IL-12 in vivo. An individualised, stepwise increase in the dose of Mif (125-4000 μg/kg) was needed to compensate for the progressive but transient downregulation of the inducible system. Repeated cycles of Mif induction (every 24 h for 10 days) were needed for optimal tumour eradication. However, complete protection against tumour rechallenge was seen in < 25% of the animals. The administration of oxaliplatin (5 mg/kg intraperitoneally) 3 days before starting the induction regimen achieved efficient elimination of liver metastases with a single cycle of IL-12 induction, and improved protection against tumour rechallenge. This was associated with a shift in the tumour microenvironment towards a more pro-immunogenic phenotype, with an increase in the CD8+/T regulatory cell ratio and a reduction in myeloid-derived suppressor cells. These effects were not seen with 5-fluorouracil, irinotecan or gemcitabine

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

Mouse Models of Peritoneal Carcinomatosis to Develop Clinical Applications

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. 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

Intratumoral virotherapy with 4-1BBL armed modified vaccinia Ankara eradicates solid tumors and promotes protective immune memory

Background Human cancers are extraordinarily heterogeneous in terms of tumor antigen expression, immune infiltration and composition. A common feature, however, is the host ' s inability to mount potent immune responses that prevent tumor growth effectively. Often, naturally primed CD8(+) T cells against solid tumors lack adequate stimulation and efficient tumor tissue penetration due to an immune hostile tumor microenvironment. Methods To address these shortcomings, we cloned tumor-associated antigens (TAA) and the immune-stimulatory ligand 4-1BBL into the genome of modified vaccinia Ankara (MVA) for intratumoral virotherapy. Results Local treatment with MVA-TAA-4-1BBL resulted in control of established tumors. Intratumoral injection of MVA localized mainly to the tumor with minimal leakage to the tumor-draining lymph node. In situ infection by MVA-TAA-4-1BBL triggered profound changes in the tumor microenvironment, including the induction of multiple proinflammatory molecules and immunogenic cell death. These changes led to the reactivation and expansion of antigen-experienced, tumor-specific cytotoxic CD8(+) T cells that were essential for the therapeutic antitumor effect. Strikingly, we report the induction of a systemic antitumor immune response including tumor antigen spread by local MVA-TAA-4-1BBL treatment which controlled tumor growth at distant, untreated lesions and protected against local and systemic tumor rechallenge. In all cases, 4-1BBL adjuvanted MVA was superior to MVA. Conclusion Intratumoral 4-1BBL-armed MVA immunotherapy induced a profound reactivation and expansion of potent tumor-specific CD8(+) T cells as well as favorable proinflammatory changes in the tumor microenvironment, leading to elimination of tumors and protective immunological memory

Characterization of woodchuck apolipoprotein A-I: a new tool for drug delivery and identification of altered isoforms in the woodchuck chronic hepatitis model

International audienceApolipoprotein A-I (ApoA-I) is the major protein component of high density lipoprotein (HDL) particles in serum, and participates in the reverse transport of cholesterol from tissues to the liver for excretion. The natural HDL tropism to the liver and cancer cells has been used extensively to target encapsulated drugs. The alteration of the plasmatic isoforms of ApoA-I is a hallmark of chronic hepatitis and hepatocarcinoma in mice and humans. Woodchucks infected with the woodchuck hepatitis virus (WHV) represent the best animal model for the study of chronic viral hepatitis B and viral induced hepatocarcinoma (HCC). WHV-infected woodchuck represents a clinically relevant animal model under which new treatment strategies can be evaluated and optimized. Therapeutic efficacy in this model is likely to be translated into a successful therapy for patients infected with HBV. The present study describes, for the first time, the cloning and characterization of woodchuck ApoA-I. The open reading frame (ORF) of the woodchuck ApoA-I is 795 bp long, coding for 264 amino acids. Unexpectedly, phylogenetic analysis revealed that the closest sequences are those of human and macaque. Woodchuck HDLs were isolated successfully from sera by density gradient ultracentrifugation. A commercial antibody that recognized the woodchuck ApoA-I was also identified. Finally, taking advantage of the techniques and tools developed in this study, two potential applications of woodchuck HDLs are illustrated: drug delivery to a woodchuck hepatocarcinoma cell line and the use of isoelectrofocusing to identify ApoA-I isoforms

Tumor Induced Hepatic Myeloid Derived Suppressor Cells Can Cause Moderate Liver Damage

<div><p>Subcutaneous tumors induce the accumulation of myeloid derived suppressor cells (MDSC) not only in blood and spleens, but also in livers of these animals. Unexpectedly, we observed a moderate increase in serum transaminases in mice with EL4 subcutaneous tumors, which prompted us to study the relationship of hepatic MDSC accumulation and liver injury. MDSC were the predominant immune cell population expanding in livers of all subcutaneous tumor models investigated (RIL175, B16, EL4, CT26 and BNL), while liver injury was only observed in EL4 and B16 tumor-bearing mice. Elimination of hepatic MDSC in EL4 tumor-bearing mice using low dose 5-fluorouracil (5-FU) treatment reversed transaminase elevation and adoptive transfer of hepatic MDSC from B16 tumor-bearing mice caused transaminase elevation indicating a direct MDSC mediated effect. Surprisingly, hepatic MDSC from B16 tumor-bearing mice partially lost their damage-inducing potency when transferred into mice bearing non damage-inducing RIL175 tumors. Furthermore, MDSC expansion and MDSC-mediated liver injury further increased with growing tumor burden and was associated with different cytokines including GM-CSF, VEGF, interleukin-6, CCL2 and KC, depending on the tumor model used. In contrast to previous findings, which have implicated MDSC only in protection from T cell-mediated hepatitis, we show that tumor-induced hepatic MDSC themselves can cause moderate liver damage.</p></div

Melanoma and lymphoma subcutaneous tumor-bearing mice suffer from mild liver damage.

<p>C57BL/6 and BALB/c mice bearing indicated subcutaneous tumors were sacrificed, when tumor diameter reached 15 mm. ALT (A) and AST (B) levels were analyzed in mouse serum (N≥8 mice per tumor, N≥6 naïve mice, 3 independent experiments). Naïve C57BL/6 mice (C, left image) or mice bearing B16 subcutaneous tumors (C, right image) were sacrificed, when tumor diameter reached 20 mm. TUNEL assays were performed on liver specimen (C; scale bar  = 100 µm; N = 2 mice per group, total of 5 TUNEL assays per group) and TUNEL positive cells were counted in 20 non-overlapping visual fields. Means of TUNEL positive cells per liver section were plotted (D). C, Representative examples of visual fields are shown. Data are expressed as mean ±SEM. *<i>p</i><0.05, ***<i>p</i><0.001, ****<i>p</i><0.0001 (by One-way ANOVA).</p

Increased expansion of liver damage-inducing MDSC exacerbates liver damage.

<p>Mice with different size subcutaneous tumors were analyzed for absolute numbers of hepatic MDSC (A and B), M-MDSC or (C), PMN-MDSC (D) and serum ALT levels (B–D). B–D, graphs correlate ALT levels with absolute numbers of MDSC and MDSC subsets. (N = 6–9 mice per tumor, 3 independent experiments). Data are expressed as mean ±SEM. *<i>p</i><0.05, **<i>p</i><0.01 (by two-tailed Student's <i>t</i> test).</p

Cytokine secretion profiles of different tumor models.

<p>Duplicates of tumor-conditioned media (A, N = 4–6 media samples per tumor cell line culture) or serum samples from tumor-bearing mice (B, N = 4–6 serum samples per group) were analyzed for interleukin-6, CCL-2, GM-CSF, M-CSF, KC and VEGF (A) or interleukin-6, CCL-2, KC, VEGF, IFN-γ and interleukin 10 (B). Serum samples from tumor-bearing mice were normalized to serum from naïve wild-type mice. ND  =  not detected. Data are expressed as mean ±SEM. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 (by One-way ANOVA).</p