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PURPOSE: Cryoablation is a minimally invasive procedure implemented to destroy solid tumors. It also results in the release of tumor antigens into the systemic circulation. Pre-clinical studies employing immunogenic tumor models have shown that cryoablation evokes anti-tumor immune responses. How cryoablation impacts immune responses in poorly immunogenic tumors has not been sufficiently explored. EXPERIMENTAL DESIGN: We employed bilateral, B16F10 melanoma model, devoid of strong immunogenic antigens. Cryoablation induced effector immune responses were investigated, also in combination with peritumoral STING agonist and systemic anti-PD1. Selective immune cell depletion, T-cell migration arrest, in vivo T-cell transplantation, and cryoablation vs surgical removal techniques were employed to determine the contribution of cryoablation and immunotherapies to systemic, anti-tumor, effector immune responses. RESULTS: Treatment of a tumor with cryoablation + STING agonist + anti-PD1 resulted in the rejection of unablated, contralateral tumor. Depletion studies demonstrated that the tumor rejection is essentially dependent on CD8+ T-cells. T-cell arrest in the lymph nodes had no effect on the rejection process. Splenic CD8+ T-cells isolated from cryoablation treated mice with B16F10 melanoma, upon transplantation into melanoma bearing recipients did not impact the recipient's tumor growth. Finally, comparison of cryoablation + STING agonist + anti-PD1 vs surgery + STING agonist + anti-PD1 in bilateral tumor model showed no difference in the rejection of contralateral tumor. CONCLUSION: Cryoablation does not significantly contribute to systemic, anti-tumor, effector immune responses in a B16F10 melanoma model. Cryoablation primarily performs tumor debulking, and immunotherapy functions independently of cryoablation in eliciting anti-tumor effector immune responses.
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The emergence of ablative therapies has revolutionized the treatment of inoperable solid tumors. Cryoablation stands out for its uniqueness of operation based on hypothermia, and for its ability to unleash the native tumor antigens, resulting in the generation of anti-tumor immune responses. It is not clearly understood how alterations in the rate of freeze impact the immune response outcomes. In this study, we tested fast freeze and slow freeze rates for their locoregional effectiveness and their ability to elicit immune responses in a B16F10 mouse model of melanoma. Tumor bearing mice treated with fast freeze protocol survived better than the ones treated with slow freeze protocol. Fast freeze resulted in a higher magnitude of CD4+ and CD8+ T-cell responses, and a significantly extended survival post re-challenge. Thus, fast freeze rate should be applied in any future studies employing cryoablation as an in vivo vaccination tool in conjunction with targeted immunotherapies.
Assuntos
Criocirurgia , Melanoma Experimental/cirurgia , Neoplasias Cutâneas/cirurgia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Citocinas/metabolismo , Feminino , Congelamento , Cinética , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos Endogâmicos C57BL , Células Mieloides/imunologia , Células Mieloides/metabolismo , Necrose , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Carga TumoralRESUMO
The gastrointestinal (GI) tract presents a notoriously difficult barrier for macromolecular drug delivery, especially for biologics. Herein, we demonstrate that ultrasound-stimulated phase change contrast agents (PCCAs) can transiently disrupt confluent colorectal adenocarcinoma monolayers and improve the transepithelial transport of a macromolecular model drug. With ultrasound treatment in the presence of PCCAs, we achieved a maximum of 44 ± 15% transepithelial delivery of 70-kDa fluorescein isothiocyanate-dextran, compared with negligible delivery through sham control monolayers. Among all tested rarefactional pressures (300-600 kPa), dextran delivery efficiency was consistently greatest at 300 kPa. To explore this unexpected finding, we quantified stable and inertial cavitation energy generated by various ultrasound exposure conditions. In general, lower pressures resulted in more persistent cavitation activity during the 30-s ultrasound exposures, which may explain the enhanced dextran delivery efficiency. Thus, a unique advantage of using low boiling point PCCAs for this application is that the same low-pressure pulses can be used to induce vaporization and provide maximal delivery.
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Meios de Contraste , Dextranos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Fluoresceína-5-Isotiocianato/análogos & derivados , Fármacos Gastrointestinais/administração & dosagem , Aumento da Imagem/métodos , Ultrassonografia/métodos , Células Cultivadas , Fluoresceína-5-Isotiocianato/administração & dosagem , Humanos , Técnicas In VitroRESUMO
BACKGROUND: Although metastasis is ultimately responsible for about 90% of breast cancer mortality, the vast majority of breast-cancer-related deaths are due to progressive recurrences from non-metastatic disease. Current adjuvant therapies are unable to prevent progressive recurrences for a significant fraction of patients with breast cancer. Autologous tumor cell vaccines (ATCVs) are a safe and potentially useful strategy to prevent breast cancer recurrence, in a personalized and patient-specific manner, following standard-of-care tumor resection. Given the high intra-patient and inter-patient heterogeneity in breast cancer, it is important to understand which factors influence the immunogenicity of breast tumor cells in order to maximize ATCV effectiveness. METHODS: The relative immunogenicity of two murine breast carcinomas, 4T1 and EMT6, were compared in a prophylactic vaccination-tumor challenge model. Differences in cell surface expression of antigen-presentation-related and costimulatory molecules were compared along with immunosuppressive cytokine production. CRISPR/Cas9 technology was used to modulate tumor-derived cytokine secretion. The impacts of cytokine deletion on splenomegaly, myeloid-derived suppressor cell (MDSC) accumulation and ATCV immunogenicity were assessed. RESULTS: Mice vaccinated with an EMT6 vaccine exhibited significantly greater protective immunity than mice vaccinated with a 4T1 vaccine. Hybrid vaccination studies revealed that the 4T1 vaccination induced both local and systemic immune impairments. Although there were significant differences between EMT6 and 4T1 in the expression of costimulatory molecules, major disparities in the secretion of immunosuppressive cytokines likely accounts for differences in immunogenicity between the cell lines. Ablation of one cytokine in particular, granulocyte-colony stimulating factor (G-CSF), reversed MDSC accumulation and splenomegaly in the 4T1 model. Furthermore, G-CSF inhibition enhanced the immunogenicity of a 4T1-based vaccine to the extent that all vaccinated mice developed complete protective immunity. CONCLUSIONS: Breast cancer cells that express high levels of G-CSF have the potential to diminish or abrogate the efficacy of breast cancer ATCVs. Fortunately, this study demonstrates that genetic ablation of immunosuppressive cytokines, such as G-CSF, can enhance the immunogenicity of breast cancer cell-based vaccines. Strategies that combine inhibition of immunosuppressive factors with immune stimulatory co-formulations already under development may help ATCVs reach their full potential.
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Neoplasias da Mama/terapia , Vacinas Anticâncer/imunologia , Fator Estimulador de Colônias de Granulócitos/imunologia , Imunogenicidade da Vacina , Recidiva Local de Neoplasia/prevenção & controle , Animais , Mama/patologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Sistemas CRISPR-Cas , Vacinas Anticâncer/administração & dosagem , Linhagem Celular Tumoral/imunologia , Linhagem Celular Tumoral/efeitos da radiação , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Feminino , Deleção de Genes , Fator Estimulador de Colônias de Granulócitos/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Recidiva Local de Neoplasia/imunologia , Resultado do TratamentoRESUMO
There is a critical unmet clinical need for bladder cancer immunotherapies capable of inducing durable antitumor immunity. We have shown that four intravesical treatments with a simple co-formulation of interleukin-12 and the biopolymer chitosan not only destroy orthotopic bladder tumors, but also promote a potent long-lasting systemic immune response as evidenced through tumor-specific in vitro killing assays, complete protection from rechallenge, and abscopal antitumor responses at distant non-treated tumors. This study investigates the immunological kinetics underlying these results. We show through depletion studies that CD8+ T cells are required for initial tumor rejection, but CD4+ T cells protect against rechallenge. We also show that even a single intravesical treatment can eliminate tumors in 50% of mice with 6/9 and 7/8 mice eliminating tumors after three or four treatments respectively. We then performed immunophenotyping studies to analyze shifts in immune cell populations after each treatment within the tumor itself as well as in secondary lymphoid organs. These studies demonstrated an initial infiltration of macrophages and granulocytes followed by increased CD4+ and CD8+ effector-memory cells. This was coupled with a decreased level of regulatory T cells in peripheral lymph nodes as well as decreased myeloid-derived suppressor cell infiltration in the bladder. Taken together, these data demonstrate the ability of properly delivered interleukin-12-based therapies to engage adaptive immunity within the tumor itself as well as throughout the body and strengthen the case for clinical translation of chitosan/interleukin-12 as an intravesical treatment for bladder cancer.
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Chitosan is a widely investigated biopolymer in drug and gene delivery, tissue engineering and vaccine development. However, the immune response to chitosan is not clearly understood due to contradicting results in literature regarding its immunoreactivity. Thus, in this study, we analyzed effects of various biochemical properties, namely degree of deacetylation (DDA), viscosity/polymer length and endotoxin levels, on immune responses by antigen presenting cells (APCs). Chitosan solutions from various sources were treated with mouse and human APCs (macrophages and/or dendritic cells) and the amount of tumor necrosis factor-α (TNF-α) released by the cells was used as an indicator of immunoreactivity. Our results indicate that only endotoxin content and not DDA or viscosity influenced chitosan-induced immune responses. Our data also indicate that low endotoxin chitosan (<0.01 EU/mg) ranging from 20 to 600 cP and 80% to 97% DDA is essentially inert. This study emphasizes the need for more complete characterization and purification of chitosan in preclinical studies in order for this valuable biomaterial to achieve widespread clinical application.
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Quitosana/química , Quitosana/imunologia , Animais , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/imunologia , Materiais Biocompatíveis/química , Linhagem Celular , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Técnicas de Transferência de Genes , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Tamanho da Partícula , Polímeros/química , Propriedades de Superfície , Engenharia Tecidual/métodos , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , ViscosidadeRESUMO
Bladder cancer is a highly recurrent disease in need of novel, durable treatment strategies. This study assessed the ability of an intravesical immunotherapy composed of a coformulation of the biopolymer chitosan with interleukin-12 (CS/IL-12) to induce systemic adaptive tumor-specific immunity. Intravesical CS/IL-12 immunotherapy was used to treat established orthotopic MB49 and MBT-2 bladder tumors. All mice receiving intravesical CS/IL-12 immunotherapy experienced high cure rates of orthotopic disease. To investigate the durability and extent of the resultant adaptive immune response, cured mice were rechallenged both locally (intravesically) and distally. Cured mice rejected 100 % of intravesical tumor rechallenges and 50-100 % of distant subcutaneous rechallenges in a tumor-specific manner. The ability of splenocytes from cured mice to lyse targets in a tumor-specific manner was assessed in vitro, revealing that lytic activity of splenocytes from cured mice was robust and tumor specific. Protective immunity was durable, lasting for at least 18 months after immunotherapy. In an advanced bladder cancer model, intravesical CS/IL-12 immunotherapy controlled simultaneous orthotopic and subcutaneous tumors in 70 % of treated mice. Intravesical CS/IL-12 immunotherapy creates a robust and durable tumor-specific adaptive immune response against bladder cancer. The specificity, durability, and potential of this therapy to treat both superficial and advanced disease are deserving of consideration for clinical translation.
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Quitosana/administração & dosagem , Interleucina-12/administração & dosagem , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/imunologia , Administração Intravesical , Animais , Linhagem Celular Tumoral , Quitosana/imunologia , Relação Dose-Resposta Imunológica , Feminino , Imunoterapia/métodos , Interleucina-12/imunologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Distribuição AleatóriaRESUMO
Interleukin-12 is a heterodimeric, pro-inflammatory cytokine that is a key driver of cell-mediated immunity. Clinical interest in IL-12 is significant due to its potent anti-tumor activity and efficacy in controlling certain infectious diseases such as Leishmaniasis and Listeria infection. For clinical applications, the ease of production and purification of IL-12 and the associated cost continues to be a consideration. In this context, we report a simple and effective heparin-affinity based purification of recombinant human IL-12 (hIL-12) from the serum-free supernatants of stable IL-12-transduced HEK293 cells. Fractionation of culture supernatants on heparin Sepharose columns revealed that hIL-12 elutes as a single peak in 500 mM NaCl. Coomassie staining and Western blot analysis showed that hIL-12 eluted in 500 mM NaCl is homogeneous. Purity of hIL-12 was ascertained by RP-HPLC and ESI-MS analysis, and found to be â¼98%. Western blot analysis, using monoclonal antibodies, demonstrated that the crucial inter-subunit disulfide bond linking the p35 and p40 subunits is intact in the purified hIL-12. Results of far UV circular dichroism, steady-state tryptophan fluorescence, and differential scanning calorimetry experiments suggest that purified hIL-12 is in its stable native conformation. Enzyme linked immunosorbent assays (ELISAs) and bioactivity studies demonstrate that hIL-12 is obtained in high yields (0.31±0.05 mg/mL of the culture medium) and is also fully bioactive. Isothermal titration calorimetry data show that IL-12 exhibits a moderate binding affinity (Kd(app)=69±1 µM) to heparin. The purification method described in this study is expected to provide greater impetus for research on the role of heparin in the regulation of the function of IL-12. In addition, the results of this study provide an avenue to obtain high amounts of IL-12 required for structural studies which are aimed at the development of novel IL-12-based therapeutics.
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Células HEK293/metabolismo , Interleucina-12/genética , Interleucina-12/isolamento & purificação , Sequência de Aminoácidos , Sítios de Ligação , Western Blotting , Cromatografia de Afinidade , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Heparina/metabolismo , Humanos , Interleucina-12/química , Interleucina-12/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Regulação para CimaRESUMO
Metastasis accounts for approximately 90% of breast cancer-related deaths. Therefore, novel approaches which prevent or control breast cancer metastases are of significant clinical interest. Interleukin-12 (IL-12)-based immunotherapies have shown promise in controlling metastatic disease, yet modest responses and severe toxicities due to systemic administration of IL-12 in early trials have hindered clinical application. We hypothesized that localized delivery of IL-12 co-formulated with chitosan (chitosan/IL-12) could elicit tumor-specific immunity and provide systemic protection against metastatic breast cancer while minimizing systemic toxicity. Chitosan is a biocompatible polysaccharide derived primarily from the exoskeletons of crustaceans. In a clinically relevant resection model, mice bearing spontaneously metastatic 4T1 mammary adenocarcinomas received intratumoral injections of chitosan/IL-12, or appropriate controls, prior to tumor resection. Neoadjuvant chitosan/IL-12 immunotherapy resulted in long-term tumor-free survival in 67% of mice compared to only 24% or 0% of mice treated with IL-12 alone or chitosan alone, respectively. Antitumor responses following chitosan/IL-12 treatment were durable and provided complete protection against rechallenge with 4T1, but not RENCA renal adenocarcinoma, cells. Lymphocytes from chitosan/IL-12-treated mice demonstrated robust tumor-specific lytic activity and interferon-γ production. Cell-mediated immune memory was confirmed in vivo via clinically relevant delayed-type hypersensitivity (DTH) assays. Comprehensive hematology and toxicology analyses revealed that chitosan/IL-12 induced transient, reversible leukopenia with no changes in critical organ function. Results of this study suggest that neoadjuvant chitosan/IL-12 immunotherapy prior to breast tumor resection is a promising translatable strategy capable of safely inducing to tumor-specific immunity and, in the long term, reducing breast cancer mortality due to progressive recurrences.