A novel immunogenic mouse model of melanoma for the preclinical assessment of combination targeted and immune-based therapy.
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ABSTRACT:
SUBMITTER: Lelliott EJ
PROVIDER: S-EPMC6361951 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Very late antigen-4 (VLA-4; also known as integrin α4β1) is expressed at high levels in aggressive and metastatic melanoma tumors and may provide an ideal target for imaging and targeted radiotherapy. 177Lu-DOTA-PEG4-LLP2A (177Lu-LLP2A) shows high affinity for VLA-4 and had high uptake in B16F10 mouse melanoma tumors in vivo. Here, we report efficacy studies of 177Lu-LLP2A, alone and combined with immune checkpoint inhibitors (ICI) in B16F10 tumor-bearing mice. Methods: B16F10 tumor cells (1 x 106) were implanted subcutaneously in C57BL/6 mice. After 8-10 days, the mice were randomized into eight groups (n = 8). 177Lu-LLP2A was injected i.v. on day 8 or 9 (single dose), and ICI antibodies were administered i.p. in 3 doses. Tumor growth was monitored over time via calipers. TUNEL staining for apoptosis was performed on fixed tumors. In a separate study, Cy3-LLP2A or Cy3-scramble LLP2A were injected in tumor-bearing mice, tumors were collected after 4 h post-injection, and then analyzed by flow cytometry and immunofluorescence microscopy using different immune cell markers. Results:177Lu-LLP2A alone showed comparable efficacy to combination anti-PD-1/PD-L1 + anti-CTLA-4 antibody treatment, while 177Lu-LLP2A together with combination ICI significantly enhanced survival. TUNEL staining showed the highest levels of apoptosis were found in the 177Lu-LLP2A + ICI groups. In addition to targeting tumor cells, 177Lu-LLP2A also binds immune cells in the B16F10 tumor microenvironment. Based on flow cytometry data, the tumor consists of ~77 % tumor cells/fibroblasts (CD45-CD49d+) and ~23 % immune cells (CD45+CD49d+), and immune cells express higher levels of VLA-4. Cy3-LLP2A and CD49d colocalized with macrophages (CD68), T cells (CD8, CD4), and B cells (CD19). Immunohistochemical analysis identified a significant colocalization of Cy3-LLP2A and CD68. Conclusion: Combination treatment of 177Lu-LLP2A and ICI targets both tumor cells and immune cells and has potential as a therapeutic agent in patients with metastatic melanoma.
| S-EPMC6278902 | biostudies-other
Project description:Chronic delta hepatitis, caused by hepatitis delta virus (HDV), is the most severe form of viral hepatitis, affecting at least 20 million hepatitis B virus (HBV)-infected patients worldwide. HDV/HBV co- or superinfections are major drivers for hepatocarcinogenesis. Antiviral treatments exist only for HBV and can only suppress but not cure infection. Development of more effective therapies has been impeded by the scarcity of suitable small-animal models. We created a transgenic (tg) mouse model for HDV expressing the functional receptor for HBV and HDV, the human sodium taurocholate cotransporting peptide NTCP. Both HBV and HDV entered hepatocytes in these mice in a glycoprotein-dependent manner, but one or more postentry blocks prevented HBV replication. In contrast, HDV persistently infected hNTCP tg mice coexpressing the HBV envelope, consistent with HDV dependency on the HBV surface antigen (HBsAg) for packaging and spread. In immunocompromised mice lacking functional B, T, and natural killer cells, viremia lasted at least 80 days but resolved within 14 days in immunocompetent animals, demonstrating that lymphocytes are critical for controlling HDV infection. Although acute HDV infection did not cause overt liver damage in this model, cell-intrinsic and cellular innate immune responses were induced. We further demonstrated that single and dual treatment with myrcludex B and lonafarnib efficiently suppressed viremia but failed to cure HDV infection at the doses tested. This small-animal model with inheritable susceptibility to HDV opens opportunities for studying viral pathogenesis and immune responses and for testing novel HDV therapeutics.
| S-EPMC6337727 | biostudies-literature
Project description:The B-raf gene is mutated in up to 66% of human malignant melanomas, and its protein product, BRAF kinase, is a key part of RAS-RAF-MEK-ERK (MAPK) pathway of cancer cell proliferation. BRAF-targeted therapy induces significant responses in the majority of patients, and the combination BRAF/MEK inhibitor enhances clinical efficacy, but the response to BRAF inhibitor and to BRAF/MEK inhibitor is short lived. On the other hand, treatment of melanoma with an immune checkpoint inhibitor, such as anti-PD-1, has lower response rate but the response is much more durable, lasting for years. For this reason, it was suggested that combination of BRAF/MEK and PD-1 inhibitors will significantly improve overall survival time.This paper develops a mathematical model to address the question of the correlation between BRAF/MEK inhibitor and PD-1 inhibitor in melanoma therapy. The model includes dendritic and cancer cells, CD 4+ and CD 8+ T cells, MDSC cells, interleukins IL-12, IL-2, IL-6, IL-10 and TGF- β, PD-1 and PD-L1, and the two drugs: BRAF/MEK inhibitor (with concentration γ B ) and PD-1 inhibitor (with concentration γ A ). The model is represented by a system of partial differential equations, and is used to develop an efficacy map for the combined concentrations (γ B ,γ A ). It is shown that the two drugs are positively correlated if γ B and γ A are at low doses, that is, the growth of the tumor volume decreases if either γ B or γ A is increased. On the other hand, the two drugs are antagonistic at some high doses, that is, there are zones of (γ B ,γ A ) where an increase in one of the two drugs will increase the tumor volume growth, rather than decrease it.It will be important to identify, by animal experiments or by early clinical trials, the zones of (γ B ,γ A ) where antagonism occurs, in order to avoid these zones in more advanced clinical trials.
| S-EPMC5517842 | biostudies-other
Project description:Tumor immunotherapy aims to overcome the immunosuppressive microenvironment within tumors, and various approaches have been developed. Tumor-associated T regulatory cells (Tregs) suppress the activation and expansion of tumor antigen-specific effector T cells, thus, providing a permissive environment for tumor growth. Therefore, optimal strategies need to be established to deplete tumor-infiltrated Tregs because systemic depletion of Tregs can result in reduced anti-tumor effector cells and autoimmunity. Here, to selectively deplete Tregs in tumors, we intratumorally injected anti-CD25 antibodies conjugated to Chlorin e6 (Ce6), a photosensitizer that absorbs light to generate reactive oxygen species. Local depletion of tumor-associated Tregs with photodynamic therapy (PDT) inhibited tumor growth, which was likely due to the altered tumor immune microenvironment that was characterized by increased infiltration of CD8+ effector T cells and the expression of IFN-? and CD107a, which is a cytolytic granule exocytosis marker in tumor tissues. Furthermore, PDT-induced intratumoral Treg depletion did not influence adaptive immune responses in a murine influenza infection model. Thus, our results show that intratumoral Treg-targeted PDT could specifically modulate tumor microenvironments by depleting Tregs and could be used as a novel cancer immunotherapy technique.
| S-EPMC5564577 | biostudies-literature
Project description:Melanomas harboring an activating BRAFV600 mutation account for 50% of all advanced melanomas. The approval of BRAF-targeted therapy revolutionized treatment of these patients with achievement of impressive responses. However, development of resistance to these drugs is a significant problem, and as such, duration of response remains low, with median progression free survival of around 11-15 months. Immune checkpoint blockers exploit the immune system to eradicate cancer and can produce durable disease control that results in long-term, treatment-free survival in some patients. These drugs have shown very impressive survival in patients with BRAF-mutated melanoma. Thus, there is a need to continue to utilize emerging data to achieve long-term disease control for patients with advanced melanoma. Combining targeted therapy with immune therapy may be one possible way to achieve this goal. In this review, the mechanisms of action of these two pathways, including the mechanistic basis of this combination, are summarized, along with results of completed and ongoing trials in triple therapy.
| S-EPMC9036333 | biostudies-literature
Project description:Therapeutic success of targeted therapy with BRAF inhibitors (BRAFi) for melanoma is limited by resistance development. Observations from preclinical mouse models and recent insights into the immunological effects caused by BRAFi give promise for future development of combination therapy for human melanoma. In our study, we used the transplantable D4M melanoma mouse model with the BRAFV600E mutation and concomitant PTEN loss in order to characterize alterations in tumor-infiltrating effector immune cells when tumors become resistant to BRAFi. We found that BRAFi-sensitive tumors displayed a pronounced inflammatory milieu characterized by high levels of cytokines and chemokines accompanied by an infiltration of T and NK cells. The tumor-infiltrating effector cells were activated and produced high levels of IFN-γ, TNF-α and granzyme B. When tumors became resistant and progressively grew, they reverted to a low immunogenic state similar to untreated tumors as reflected by low mRNA levels of proinflammatory cytokines and chemokines and fewer tumor-infiltrating T and NK cells. Moreover, these T and NK cells were functionally impaired in comparison to their counterparts in BRAFi-sensitive tumors. Their effector cell function could be restored by additional peritumoral treatment with the TLR7 agonist imiquimod, a clinically approved agent for nonmelanoma skin cancer. Indeed, resistance to BRAFi therapy was delayed and accompanied by high numbers of activated T and NK cells in tumors. Thus, combining BRAFi with an immune stimulating agent such as a TLR ligand could be a promising alternative approach for the treatment of melanoma.
| S-EPMC7003881 | biostudies-literature
Project description:ObjectiveTo investigate the effectiveness of combination treatment of vascular targeted photodynamic therapy and anti-cytotoxic T-lymphocyte-associated antigen 4 immunotherapy in a mouse model of urothelial carcinoma.MethodsWe used C57BL/6 mice injected with murine bladder 49 cell line. Mice were randomly allocated into four treatment groups: vascular targeted photodynamic therapy only, anti-cytotoxic T-lymphocyte-associated antigen 4 only, combination therapy and control. We carried out three separate experiments that used distinct cohorts of mice: tumor growth and development of lung metastases monitored with bioluminescent imaging (n = 91); survival evaluated with Kaplan-Meier curves (n = 111); and tumor cell population studied with flow cytometry (n = 20). In a fourth experiment, we re-challenged tumors in previously treated mice and compared tumor growth with that of naïve mice.ResultsCombination therapy provided significant benefits over the other three treatment groups: prolonged survival (P < 0.0001), lower tumor signal (P < 0.0001) and decreased lung signal uptake (P ≤ 0.002). We also observed that mice previously treated with vascular targeted photodynamic therapy only or combination therapy did not present tumor growth after re-challenged tumors.ConclusionsCombination of vascular targeted photodynamic therapy with anti-cytotoxic T-lymphocyte-associated antigen 4 is an effective therapy in a urothelial carcinoma syngeneic mouse model. The present results suggest this therapy as a potential treatment option for both bladder and upper tract tumors in future clinical trials.
| S-EPMC7583638 | biostudies-literature
Project description:Resistance to targeted therapy is an ongoing problem for the successful treatment of Stage IV metastatic melanoma. For many patients, the use of targeted therapies, such as BRAF kinase inhibitors, were initially promising yet resistance inevitably occurred. Even after combining BRAF kinase inhibitors with MEK pathway inhibitors to offset re-activation of the MAP kinase pathway, resistance is still documented. Similarly, outcomes with immune checkpoint inhibitors as monotherapy were optimistic for some patients without relapse or progression, yet the majority of patients undergoing monotherapy have progressive disease. Will immunotherapy and combination therapy trials overcome resistance in metastatic melanoma? In an effort to treat resistant disease, new clinical trials evaluating the combination of immunotherapy with other therapies, such as kinase inhibitors, adoptive cell therapy, chimeric CD40 ligand to boost costimulation, or a tumor-specific oncolytic virus enhancing granulocyte macrophage colony-stimulating factor (GM-CSF) expression, are currently underway. Updated studies on the mechanisms of resistance, immune escape and options to reinvigorate immune cells support the continued discovery of new and improved forms of therapy.
| S-EPMC5650456 | biostudies-literature
Project description:PurposeUveal melanoma (UM) is considered a rare disease; yet, it is the most common intraocular malignancy in adults. Although the primary tumor may be efficiently managed, more than 50% of patients with UM develop distant metastases. The mortality at the first year after diagnosis of metastatic UM has been estimated at 81%, and the poor prognosis has not improved in the past years due to the lack of effective therapies.MethodsIn order to search for novel therapeutic possibilities for metastatic UM, we performed a small-scale screen of targeted drug combinations. We verified the targets of the tested compounds by western blotting and PCR and clarified the mechanism of action of the selected combinations by caspase 3 and 7 activity assay and flow cytometry. The best two combinations were tested in a mouse patient-derived xenograft (PDX) UM model as putative therapeutics for metastatic UM.ResultsCombinations of the multitarget drug trabectedin with either the CK2/CLK double-inhibitor CX-4945 (silmitasertib) or the c-MET/TAM (TYRO3, Axl, MERTK) receptor inhibitors foretinib and cabozantinib demonstrated synergistic effects and induced apoptosis (relative caspase 3 and 7 activity increased up to 20.5-fold in UM cell lines). In the case of the combination of foretinib and cabozantinib, inhibition of the TAM receptors, but not c-Met, was essential to inhibit the growth of UM cells. Monotreatment with trabectedin inhibited tumor growth by 42%, 49%, and 35% in the MM26, MM309, and MM339 PDX mouse models, respectively.ConclusionsTrabectedin alone or in combination with cabozantinib inhibited tumor growth in PDX UM mouse models. Blocking of MERTK, rather than TYRO3, activity inhibited UM cell growth and synergized with trabectedin.
| S-EPMC9756579 | biostudies-literature
Project description:Malignant melanoma is an aggressive type of skin cancer whose incidence is increasing globally. Although surgery is effective in early stage melanoma, patients with advanced melanoma only have a 20% 5-year survival rate. Hence, combinations of existing and new immunotherapy technologies and immunotherapeutic agents are being evaluated. ONCOS-102 is an oncolytic adenovirus armed with human GM-CSF and an Ad5/3 chimeric capsid. It has shown to be well tolerated in phase I study (NCT01598129) wherein it induced antitumor immunity, infiltration of CD8 + T cells to tumors, and up-regulation of PD-L1. We propose that ONCOS-102 could serve as an immunosensitizer in combination therapies with checkpoint inhibitors. In this preclinical study, we investigated the cytotoxicity of ONCOS-102 and pembrolizumab, an anti-PD-1 antibody, in four human melanoma cell lines, A375, A2058, SK-Mel-2 and SK-Mel-28. Humanized mice engrafted with A2058 melanoma cells showed significant tumor volume reduction after ONCOS-102 treatment. Combination of pembrolizumab with ONCOS-102 reduced tumor volume to an even greater extent, while pembrolizumab (200 µg, or 400 µg) did not show any therapeutic benefit by itself. Body weight loss, and metastasis were not significantly affected by any treatment. These data support the scientific rationale for the ongoing clinical study of combination therapy of ONCOS-102 and pembrolizumab for the treatment of melanoma (NCT03003676).
| S-EPMC6343802 | biostudies-literature