Irradiated Melanoma Cells Research Articles

Phase II trial of vaccination with autologous, irradiated melanoma cells engineered by adenoviral mediated gene transfer to secrete granulocyte-macrophage colony stimulating factor in patients with stage III and IV melanoma.

In the era of immune checkpoint blockade, the role of cancer vaccines in immune priming has provided additional potential for therapeutic improvements. Prior studies have demonstrated delayed type hypersensitivity and anti-tumor immunity with vaccines engineered to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF). The safety, efficacy and anti-tumor immunity of GM-CSF secreting vaccine in patients with previously treated stage III or IV melanoma needs further investigation. In this phase II trial, excised lymph node metastases were processed to single cells, transduced with an adenoviral vector encoding GM-CSF, irradiated, and cryopreserved. Individual vaccines were composed of 1x106, 4x106, or 1x107 tumor cells, and were injected intradermally and subcutaneously at weekly and biweekly intervals. The primary endpoints were feasibility of producing vaccine in stage III patients and determining the proportion of patients alive at two years in stage IV patients. GM-CSF vaccine was successfully developed and administered in all 61 patients. Toxicities were restricted to grade 1-2 local skin reactions. The median OS for stage III patients (n = 20) was 71.1 (95% CI, 43.7 to NR) months and 14.9 (95%CI, 12.1 to 39.7) months for stage IV patients. The median PFS in stage III patients was 50.7 (95%CI, 36.3 to NR) months and 4.1 (95% CI, 3.0-6.3) months in stage IV patients. In the overall population, the disease control rate was 39.3% (95%CI, 27.1 to 52.7%). In stage III patients, higher pre-treatment plasma cytokine levels of MMP-1, TRAIL, CXCL-11, CXCL-13 were associated with improved PFS (p<0.05 for all). An increase in post-vaccination levels of IL-15 and TRAIL for stage III patients was associated with improved PFS (p=0.03 for both). Similarly, an increase in post-vaccination IL-16 level for stage IV patients was associated with improved PFS (p=0.02) and clinical benefit. Vaccination with autologous melanoma cells secreting GM-CSF augments antitumor immunity in stage III and IV patients with melanoma, is safe, and demonstrates disease control. Luminex data suggests that changes in inflammatory cytokines and immune cell infiltration promote tumor antigen presentation and subsequent tumor cell destruction. Additional investigation to administer this vaccine in combination with immune checkpoint inhibitors is needed.

Differences in the phospholipid profile of melanocytes and melanoma cells irradiated with UVA and treated with cannabigerol and cannabidiol

UV radiation inducing mutations in melanocytes might cause melanoma. As changes in lipid composition and metabolism are associated with many types of cancer including skin cancer, we aimed to evaluate the effects of two phytocannabinoids cannabidiol (CBD) and cannabigerol (CBG), on changes in phospholipid and ceramide (CER) profiles induced by UVA irradiation in human melanocytes and melanoma. UVA radiation caused a significant up-regulation PC, PI and SM species and decrease of CERs content in both types of cells, while up-regulation of PEo was only observed in melanocytes. Exposure of UVA-irradiated melanocytes or melanoma cells to CBD and/or CBG led to significant decrease in relative content of PC, PI and SM specie; however, this effect was more pronounced in cancer cells. Interestingly, only in UVA-irradiated melanocytes and not in melanoma, PEo content was lowered after CBD treatment, while CBG led to additional up-regulation of PEo species. CBD and CBG used together caused decrease of zeta potential, inhibiting PS externalization, and different changes in relative contents of CER and SM species of irradiated and non-irradiated melanoma cells. Obtained results are quite promising due to CBD and CBG abilities to partial reverse pro-cancerogenic changes in phospholipid and CER profiles induced by UVA.

Cellular antioxidant and cytotoxic activity of astaxanthin and ellagic acid on UV irradiated skin melanoma cells and gel formulation

Ultraviolet radiation accounts for most non-melanoma skin cancers (carcinoma of basal and squamous cells) and melanoma skin cancers. Hence, UV radiation is considered one of the most potent physical carcinogens. Radiation exposure leads to water hydrolysis and heightens free radical production. The free radicals, in turn, react with macromolecules such as proteins, DNA, RNA, and lipids and alters their structure. Antioxidants protect the cells against radiation-induced cell death and DNA damage. It was also attributed to the reduction in ROS induced by UV radiation. Both ellagic acid and astaxanthin has antioxidant property. In addition to the antioxidant property, these compounds possess inflammatory, anti-proliferative, and wound-healing properties. The study aims to evaluate the ROS scavenging potential of ellagic acid and astaxanthin in Skin melanoma (SK-Mel-28) cells and formulate a gel that can treat radiation dermatitis. The spectral characterization of ellagic acid and astaxanthin was carried out using UV–Vis spectroscopy and FTIR. The antioxidant potential of astaxanthin and ellagic acid was measured using probe 2′, 7′-dichlorofluorescein-diacetate in UV-treated skin melanoma cells. These antioxidants attenuated the intracellular ROS in UV-treated SK-Mel-28 cells, evident at 25 μg/ml and 50 μg/ml. Comparative fluorescent intensity profile analyzed using ImageJ also showed that both astaxanthin and ellagic acid exhibit high ROS scavenging potential without prooxidant capacity. The ellagic acid extract and astaxanthin decreasedSK-Mel-28 cell viability with a median inhibitory concentration IC50 of 47.23 μg/ml and 41.39 μg/ml. The ellagic acid enhanced thiobarbituric acid reactive substance level, an indicator of lipid peroxidation with a median stimulatory concentration EC50 of 53.7 μg/ml in SK-Mel-28 cells. This substantiated that ellagic acid potentiates the antitumor effect through lipid peroxidation. It was proposed that the astaxanthin-ellagic acid gel formulation produced better results in reducing UV-induced skin cancers due to positive synergism.

Photosensitizer‐Free Phototherapy with Peptide Micelle Nanoadjuvants for Cancer Vaccine against Metastasis of Melanoma

AbstractHere, an external photosensitizer‐free vaccination strategy accompanied by delivery of nanoadjuvants is reported as a treatment for melanoma. Melanin‐enriched B16F10 melanoma cells showed a strong and specific photothermal effect under irradiation of near‐infrared (NIR) wavelength light, and a corresponding killing effect of cancer cells is observed. The irradiated melanoma cells exhibited surface exposure of the eat‐me signal, calreticulin, which induced immunogenic cell death. The nanoadjuvant, which comprised imiquimod encapsulated in amphiphilic peptide‐based micelles, induced dendritic cell maturation. In B16F10 melanoma tumor‐bearing mice, irradiation of NIR light alone increased the temperature of the tumor site to 60 °C regardless of nanoadjuvant treatment. To mimic metastasis to sites near the primary tumor site, primary tumor‐cured mice are rechallenged with B16F10 cells. In a lung metastasis model induced by intravenous injection of B16F10 cells, only nanoadjuvant‐treated group showed significant prevention of B16F10 tumor nodule formation in the lung. The immunotherapeutic effects of this nanoadjuvant are supported by an observed increase of tumor‐infiltrating CD8 + T cell populations. The results suggest that the combination of photosensitizer‐free phototherapy with a peptide micelle nanoadjuvant has strong potential for clinical translation as a melanoma vaccine.

Abstract 1072: Lymphangiogenesis-inducing vaccines for melanoma treatment elicit potent tumor-specific T cell immunity and long-term tumor control

Abstract We recently demonstrated that the growth of tumor-associated lymphatic vessels (lymphangiogenesis) promotes immune infiltration in melanoma (Lund et al. 2016) and increases T cell activation following immunotherapy, resulting in strongly enhanced therapeutic efficacy (Fankhauser et a. 2017). On the other hand, tumor lymphangiogenesis is known to support cancer cell dissemination through the lymphatic route and metastasis formation. Therefore, inducing lymphangiogesis within the tumor site to boost anti-tumor immunity appears as a potentially unsafe therapeutic approach since it might also result in increased tumor metastatic potential. In this study, we sought to exploit the immune-promoting functions of lymphatics remotely from the tumor by developing a lymphangiogenesis-inducing cancer vaccine that mimics the microenvironment of a lymphangiogenic tumor. Whole-cell lymphangiogenic vaccines were generated using lethally irradiated mouse melanoma cells genetically engineered to overexpress the lymphatic-specific growth factor VEGFC, in combination with topical immune adjuvants (VEGFC vax). VEGFC vax was compared to control vaccines with identical formulation but lacking VEGFC overexpression and to a vaccine composed of GM-CSF-overexpressing tumor cells (GVAX), a vaccination approach that has been widely used in clinical studies. Upon intradermal injection in mice, VEGFC vax induced extensive lymphangiogenesis at the vaccine site and increased antigen transport to vaccine-draining lymph nodes. Analogously to what observed in lymphangiogenic tumors, we found that naïve T cells significantly infiltrated lymphangiogenic vaccine sites and could undergo in situ priming and activation. Using VEGFC vaccines containing either B16-F10 mouse melanoma cells or melanoma cells obtained from tumors growing in BrafV600EPten−/- transgenic mice, we demonstrated that VEGFC vax elicits a potent tumor-specific T cell response. Importantly, VEGFC vaccination resulted in the mounting of a broad T cell immunity directed against multiple B16-F10-associated antigens. In both prophylactic and therapeutic settings, VEGFC vax provided effective tumor control and long-term protection against B16-F10 melanomas. In the present study we introduced for the first time the concept of lymphangenesis induction as a tool to increase cancer vaccine potency and we provided a proof of efficacy of lymphangiogenic vaccines in preclinical melanoma models. Citation Format: Maria Stella Sasso, Nikolaos Mitrousis, Sylvie Hauert, Priscilla S. Briquez, Yue Wang, Jun Ishihara, Jeffrey A. Hubbell, Melody A. Swartz. Lymphangiogenesis-inducing vaccines for melanoma treatment elicit potent tumor-specific T cell immunity and long-term tumor control [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1072.

The Highlighted Role of GAPDH and Nitric-Oxide Synthase Regulator Activity in Proton Beam Irradiated Melanoma BLM Cells.

Introduction: The human melanoma is a type of invasive tumor the treatment of which is challenging. To better understand the proton irradiation mechanisms as one of the widely applied therapy for this type of cancer, bioinformatics analysis of proteomics outcome could be beneficial. Methods: Protein-protein interaction network analysis of the differentially expressed proteins (DEPs) of melanoma BLM (BRO lung metastasis) cells in the treatment of 3 Gy dosage proton therapy was performed in this study via Cytoscape V.3.7.2. and its integrated plug-ins. Results: Eighteen DEPs were searched for network constructions and limited numbers of query +neighbor proteins were found central. The hub-bottlenecks (i.e. central nodes) were GAPDH, ACTB, ALB, AKT1, TP53, and EGFR. The fist mentioned proteins were from DEPs. The enrichment analysis of these elements identified nitric-oxide synthase regulator activity and the positive regulation of the norepinephrine uptake that may be the key to the mechanisms of proton therapy. Conclusion: In conclusion, the identified central nodes (EGFR, TP53, ALB, AKT1, GAPDH, and ACTB) and the related biological terms are the critical affected genes and biological terms in the irradiated melanoma cells.

Antisense targeting of CD47 enhances human cytotoxic T-cell activity and increases survival of micebearing B16 melanoma when combined with anti-CTLA4 and tumor irradiation.

Antibodies targeting the T-cell immune checkpoint cytotoxic T-lymphocyte antigen-4 (CTLA4) enhance the effectiveness of radiotherapy for melanoma patients, but many remain resistant. To further improve response rates, we explored combining anti-CTLA4 blockade with antisense suppression of CD47, an inhibitory receptor on T cells that limit T-cell receptor signaling and killing of irradiated target cells. Human melanoma data from The Cancer Genome Atlas revealed positive correlations between CD47 mRNA expression and expression of T-cell regulators including CTLA4 and its counter receptors CD80 and CD86. Antisense suppression of CD47 on human T cells in vitro using a translational blocking morpholino (CD47m) alone or combined with anti-CTLA4 enhanced antigen-dependent killing of irradiated melanoma cells. Correspondingly, the treatment of locally irradiated B16F10 melanomas in C57BL/6 mice using combined blockade of CD47 and CTLA4 significantly increased the survival of mice relative to either treatment alone. CD47m alone or in combination with anti-CTLA4 increased CD3+ T-cell infiltration in irradiated tumors. Anti-CTLA4 also increased CD3+ and CD8+ T-cell infiltration as well as markers of NK cells in non-irradiated tumors. Anti-CTLA4 combined with CD47m resulted in the greatest increase in intratumoral granzyme B, interferon-γ, and NK-cell marker mRNA expression. These data suggest that combining CTLA4 and CD47 blockade could provide a survival benefit by enhancing adaptive T- and NK-cell immunity in irradiated tumors.

Syngeneic B16-F1 cells are more efficient than allogeneic Cloudman cells as antigen source in DC-based vaccination in the B16-F1 murine melanoma model

A major obstacle to obtaining relevant results in cancer vaccination has been the lack of identification of immunogenic antigens. Dendritic cell (DC)-based cancer vaccines used preventively may afford protection against tumor inoculation, but the effect of antigen choice on anti-tumor protection is not clear. When using irradiated syngeneic tumor cells to load DCs, tumor self-antigens are provided, including tumor-associated antigens (TAAs) and neoantigens generated by tumor mutations. On the other hand, allogeneic tumor cells could only supply shared TAAs. To assess the advantages of each source in protective vaccination, we analyzed in C57BL/6 mice the effect of loading DCs with irradiated syngeneic B16-F1 or allogeneic Cloudman melanoma cells; both cell lines were characterized by whole exome sequencing and RNAseq. Tumor cell components from the two irradiated cell lines were efficiently internalized by DCs, and transported to MHC-class II positive tubulovesicular compartments (MIICs). DCs loaded with allogeneic irradiated Cloudman cells (DC-ApoNecALLO) induced a partially effective anti-melanoma protection, although Cloudman and B16-F1 cells share the expression of melanocyte differentiation antigens (MDAs), cancer-testis antigens (CTAs) and other TAAs. DCs loaded with syngeneic B16-F1 cells (DC-ApoNecSYN) established a more potent and long-lasting protection and induced a humoral anti-B16F1 response, thus suggesting that neoepitopes are needed for inducing long-lasting protection.

Abstract 1448: Lymphangiogenesis-inducing whole-cell vaccine against melanoma promotes broad tumor-specific T cell immunity and effective tumor control

Abstract We recently reported that the growth of lymphatic vessels (lymphangiogenesis) in mouse melanomas dramatically enhances the efficacy of immunotherapy by promoting immune cell infiltration and broadening the anti-tumor T cell repertoire (Fankhauser et al, Sci. Trans. Med. 2017). Correlative evidence supporting such ‘lymphangiogenic potentiation’ was also seen in human melanoma patients, suggesting a possible immunotherapeutic use of the pro-lymphangiogenic growth factor VEGFC. On the other hand, tumor lymphangiogenesis is known to promote cancer cell dissemination and metastasis, raising concerns about the possibility of manipulating lymphatic vessels directly within the tumor site as a treatment strategy for cancer immunotherapy. Here, we sought to manipulate and exploit the immune-promoting functions of lymphatics remotely from the tumor by developing a lymphangiogenesis-inducing cancer vaccine that mimics the microenvironment of a lymphangiogenic tumor. Whole-cell vaccines were formulated using lethally irradiated B16F10 or B16F10-OVA mouse melanoma cells, either mock-transduced or genetically modified to overexpress VEGFC, and combined with topically retained immune adjuvants (ctrl-vax and VEGFC-vax, respectively). Upon intradermal injection, VEGFC-vax induced extensive local lymphatic growth and stimulated increased lymphatic transport from the vaccine site to the draining lymph nodes. Consistently with our previous study, VEGFC induced CCL21 upregulation and local recruitment of CCR7+ naïve T cells, which could undergo in situ priming in the vaccine site. Compared to ctrl-vax, VEGFC-vax elicited a robust T cell response directed against multiple mouse melanoma antigens, as assessed by ex vivo antigen stimulation and IFNγ ELISPOT. In a prophylactic vaccine setting, VEGFC-vax induced stronger tumor-specific T cell immunity and greater protection from tumor challenge compared to both ctrl-vax as well as a GM-CSF-secreting whole-cell vaccine. Overall this study provides a proof of concept for the use of VEGFC in cancer vaccines and characterizes a whole-cell lymphangiogenesis-inducing vaccine formulation with potential for clinical translation. Citation Format: Maria Stella Sasso, Sylvie Hauert, Priscilla S. Briquez, Yue Wang, Jun Ishihara, Jeffrey A. Hubbell, Melody A. Swartz. Lymphangiogenesis-inducing whole-cell vaccine against melanoma promotes broad tumor-specific T cell immunity and effective tumor control [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1448.

Overexpression of Ras-Related C3 Botulinum Toxin Substrate 2 Radiosensitizes Melanoma Cells In Vitro and In Vivo.

Radioresistance is the major obstacle in the radiotherapy of the malignant melanoma. Thus, it is of importance to increase the radiosensitivity of melanoma cells. In the present study, the radioresistant melanoma cell line OCM-1 with inducible overexpression of Ras-related C3 botulinum toxin substrate 2 was established based on a radiation-inducible early growth response gene (Egr-1) promoter. The effects of Ras-related C3 botulinum toxin substrate 2 overexpression on the radiosensitivity of melanoma cells exposed to either X-rays or carbon ion beams were evaluated in cultured cells as well as xenograft tumor models. In addition, both reactive oxygen species yield and the NADPH oxidase activity were measured in the irradiated melanoma cells. It was found that the radiation-inducible overexpression of Ras-related C3 botulinum toxin substrate 2 sensitized the melanoma cells to both X-rays and carbon ion irradiation by enhancing the NADPH oxidase activity and the subsequent reactive oxygen species production. Besides, the overexpression of Ras-related C3 botulinum toxin substrate 2 enhanced the tumor-killing effect of radiotherapy in xenograft tumors significantly. The results of this study indicate that Ras-related C3 botulinum toxin substrate 2 is promising in increasing the radiosensitivity of melanoma cells, which provides experimental evidence and theoretical basis for clinical radiosensitization of the malignant melanoma.

Abstract 2984: Effects of anti-CTLA-4 and anti-PD-1 on memory T-cell differentiation and resistance to tumor relapse

Abstract The FDA has begun to expand the approved uses of immune checkpoint blockade antibodies targeting CTLA-4 and PD-1. Blocking either checkpoint relieves the negative regulation of T-cells resulting in significant responses in patients with cancer. Data has now begun to emerge regarding differences between these two therapies. While αPD-1 therapy has a greater response rate (~30% vs. 11%) according to RECIST criteria, recent reports have suggested responses to αPD-1 may not be as durable as αCTLA-4. 25% of patients who initially responded to αPD-1 have tumor relapse within 24 months. In contrast, the 3-year survival rate of patients treated with αCTLA-4 is at least ~25% suggesting a durable response. Previous studies in bacterial or chronic LCMV infectious models have shown that αCTLA-4 can increase CD8+ memory T-cell formation, whereas genetic ablation of PD-1 on T-cells often promotes the terminally differentiated exhausted CD8+ T-cell phenotype, while attenuating memory T-cell formation. However, the mechanism which leads to relapse following αPD-1 treatment in tumor models is not clear. The goal of this project is to understand how immunotherapies shape memory T-cell formation and how that relates to the mechanism of tumor relapse. To test whether αCTLA-4 or αPD-1 can induce a better memory T-cell response, mice were vaccinated with irradiated B16F10 murine melanoma cells and treated αCTLA-4 or αPD-1. Mice were re-challenged with B16F10 80 days after vaccination. Although both αCTLA-4 and αPD-1 improved tumor rejection compared with controls, αCTLA-4 treated mice exhibited superior tumor control compared to αPD-1 (p&amp;lt;0.0005) suggesting the memory T-cell response mediated by αCTLA-4 is more durable. In order to test whether this memory T-cell response is antigen-specific, mice were re-challenged with unrelated MC38 or 3LL cancer cells. The antibody treated groups did not show improved antitumor effect compared with vaccine control (p&amp;gt;0.5). To test whether the frequency of memory T-cells recruited to the re-challenged tumor could affect memory T-cell response, antigen-specific pmel-1 T-cells were infused to mice following vaccination with αCTLA-4 or αPD-1. Our result suggested that there were more tumor-infiltrating pmel-1 T-cells in the αCTLA-4 treated group compared to the αPD-1 treated group (p&amp;lt;0.05). In order to augment the durability of αPD-1 treatment, αPD-1 was combined with αCTLA-4 following vaccination. The combined treatment group has superior antitumor response compared to that with αPD-1 (p&amp;lt;0.000003) and overlapped with the αCTLA-4 treated group (P&amp;gt;0.05) during re-challenge, indicating that the effect of the combined treatment is dominated by αCTLA-4. Collectively, our studies facilitate the design of combination immunotherapy treatments that enhance both response rates and generation of memory T-cells to prevent relapse. Citation Format: Stephen Mok, Colm R. Duffy, James P. Allison. Effects of anti-CTLA-4 and anti-PD-1 on memory T-cell differentiation and resistance to tumor relapse [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 2984.

Three-dimensional experiments and individual based simulations show that cell proliferation drives melanoma nest formation in human skin tissue

BackgroundMelanoma can be diagnosed by identifying nests of cells on the skin surface. Understanding the processes that drive nest formation is important as these processes could be potential targets for new cancer drugs. Cell proliferation and cell migration are two potential mechanisms that could conceivably drive melanoma nest formation. However, it is unclear which one of these two putative mechanisms plays a dominant role in driving nest formation.ResultsWe use a suite of three-dimensional (3D) experiments in human skin tissue and a parallel series of 3D individual-based simulations to explore whether cell migration or cell proliferation plays a dominant role in nest formation. In the experiments we measure nest formation in populations of irradiated (non-proliferative) and non-irradiated (proliferative) melanoma cells, cultured together with primary keratinocyte and fibroblast cells on a 3D experimental human skin model. Results show that nest size depends on initial cell number and is driven primarily by cell proliferation rather than cell migration.ConclusionsNest size depends on cell number, and is driven primarily by cell proliferation rather than cell migration. All experimental results are consistent with simulation data from a 3D individual based model (IBM) of cell migration and cell proliferation.

Modulation by neighboring cells of the responses and fate of melanoma cells irradiated with UVA

UVA radiation, which accounts for about 95% of the solar spectrum, contributes to and may be the etiological factor of skin cancers of which malignant melanoma is the most aggressive. UVA causes oxidative stress in various types of cells in the skin, keratinocyte, melanocytes, and fibroblasts, which is responsible for its cytotoxic effect. Here we used a transwell system to explore how the responses of melanoma cells to a low dose of UVA (20kJ/m2, ~10% of the minimal erythema dose) are influenced by neighboring co-cultured melanoma cells or fibroblasts. This dose had a low toxicity for melanoma cells, but after irradiation, co-culture with non-irradiated melanoma cells caused a strong decline in their viability and an increased frequency of apoptosis, whereas co-culture with fibroblast exerted a protective effect on irradiated melanoma cells. At the same time, the presence of non-irradiated cells, especially fibroblasts, decreased the level of UVA-induced reactive oxygen and nitrogen species. Interleukins efficiently produced by fibroblasts seem to be main players in these effects. Our studies reveal that coexistence of fibroblasts with melanoma cells may strongly modulate the direct action and may change bystander effects exerted by UVA light. Similar modulation of the effect of UVA on melanoma cells in vivo by bystander-like signaling from neighboring cells would have consequences for the development of malignant melanoma.

Upregulation of caprin1 expression is associated with poor prognosis in hepatocellular carcinoma

Caprin-1 has been found to promotes osteosarcoma tumor growth, lung metastasis in mice, regulating the proliferation and invasion of human breast cancer cells and up-regulated in proton beam irradiated human melanoma cells. However, its clinical role, biological function in the hepatocellular carcinoma (HCC) remains unknown. In the present study, we investigated the clinical significance of Caprin1 in the HCC. Caprin1 expression was detected by immunohistochemistry, and the expression level was analyzed in 65 HCC tissues and paired peritumoral tissue. Prognostic value of Caprin1 in HCC was evaluated in 174 cases using Kaplan-Meier analysis. Univariate survival analysis and multiple Cox proportional hazards regression were performed using the Cox regression analysis. Immunohistochemistry revealed that Caprin1 expression in 65 HCC tissues was upregulated compared to paired peritumoral tissue (p=0.0064). Survival analysis in 174 HCC patient tissues was showed that high Caprin1 expression was significantly associated with worse overall survival (HR=1.513, p=0.042). In conclusion, high Caprin1 expression independently predicts a poor outcome for patients with HCC, supporting that Caprin1 may be a promising novel HCC prognostic factor.

Abstract 3997: Interleukin-4 receptor-targeted cytotoxic T cells enhances the therapeutic efficacy of adoptive T cell therapy against melanoma

Abstract Melanoma, the deadliest form of skin cancer, has long been a major focus of immunotherapy. Adoptive T cell therapy-based cancer immunotherapy has been used to enhance the specificity and potential of host immune system to treat melanoma. However, the infiltration and antitumor function of cytotoxic T cells (CTLs) are blocked or down-regulated by various signals derived from tumor cells and cells in tumor microenvironment. A novel approach to specify and enhance the homing ability of CTLs to tumor is of high demand. Interleukin-4 receptor (IL-4R) is over-expressed on many types of cancer cells, including melanoma, and has been widely employed for targeted drug delivery. In this work, we examined whether the tumor homing efficiency and anti-tumor therapeutic efficacy of CTLs could be heighten by labeling CTLs with IL4RPep-1, a targeting peptide that binds to IL-4R. The labeling was empowered by conjugating IL4RPep-1 with Dioleylphosphatidylethanolamine-biological anchor for membrane (DOPE-BAM), an oleyl acid-derived membrane anchor that incorporates into lipid membrane of cells. Tumor-specific CTLs isolated from the mice immunized by irradiated B16F10 melanoma cells were highly populated with CD62L+CD44+ activated T cells. After labeling with IL4RPep-1 via DOPE-BAM, CTLs were still functionally active, as determined by intracellular interferon-γ and granzyme release assays. Moreover, such labeling did not affect the proliferation of T cells. Adoptive transfer of IL4RPep-1-labeled, IL-4R-targeted CD45.1+CTLs into CD45.2 mice bearing B16F10 melanoma enhanced their accumulation at tumor compared to that of unlabeled T cells. Tumor homing of IL4RPep-1-labeled T cells was further validated by in vivo fluorescence imaging of mice bearing 4T1 tumor at mammary gland. Adoptive transfer of IL-4R-targeted CTLs showed remarkable anti-tumor growth activity in mice bearing B16F10 tumor compared to untargeted CTLs. After the transfer, flow cytometric study of immune cells showed significant increase of CD8+ T cells and F4/80+CD86+M1-polarized macrophages with noticeable decrease in tumor-promoting immune cells, such as F4/80+CD206+M2-polarized macrophages, Gr1+CD11b+ myeloid derived suppressor cells, and CD3+CD4+ T cells. The CTLs accumulated at tumor were mostly exogenous CD45.1+ T cells. These results implicate that IL-4R-targeted CTLs, which is enabled by labeling of with IL4RPep-1, can be a promising strategy in the field of adoptive T cell therapy against IL-4R-overexpressing tumor. Citation Format: Gunassekaran Gowri Rangaswamy, Poongkavithai Vadevoo Sri Murugan, Guruprasath Padmanaban, Lianhua Chi, Ha-Jeong Kim, Byung Heon Lee. Interleukin-4 receptor-targeted cytotoxic T cells enhances the therapeutic efficacy of adoptive T cell therapy against melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3997. doi:10.1158/1538-7445.AM2017-3997

Abstract 1685: Effects of anti-ctla-4 and anti-pd-1 on memory T-cell differentiation and resistance to tumor relapse

Abstract The FDA has begun to expand the approved uses of immune checkpoint blockade antibodies targeting CTLA-4 and PD-1. Blocking either checkpoint relieves the negative regulation of T-cells resulting in significant responses in patients with cancer. Data has now begun to emerge regarding differences between these two therapies. While αPD-1 therapy has a greater response rate (~30% vs. 11%) according to RECIST criteria, recent reports have suggested responses to αPD-1 may not be as durable as αCTLA-4. 25% of patients who initially responded to αPD-1 have tumor relapse within 24 months. In contrast, the 3-year survival rate of patients treated with αCTLA-4 is at least ~25% suggesting a durable response. Previous studies in bacterial or chronic LCMV infectious models have shown that αCTLA-4 can increase CD8+ memory T-cell formation, whereas genetic ablation of PD-1 on T-cells often promotes the terminally differentiated exhausted CD8+ T-cell phenotype, while attenuating memory T-cell formation. However, the mechanism which leads to relapse following αPD-1 treatment in tumor models is not clear. The goal of this project is to understand how immunotherapies shape memory T-cell formation and how that relates to the mechanism of tumor relapse. To test whether αCTLA-4 or αPD-1 can induce a better memory T-cell response, mice were vaccinated with irradiated B16F10 murine melanoma cells and treated αCTLA-4 or αPD-1. Mice were re-challenged with B16F10 80 days after vaccination. Although both αCTLA-4 and αPD-1 improved tumor rejection compared with controls, αCTLA-4 treated mice exhibited superior tumor control compared to αPD-1 (p&amp;lt;0.0005) suggesting the memory T-cell response mediated by αCTLA-4 is more durable. In order to test whether this memory T-cell response is antigen-specific, mice were re-challenged with unrelated MC38 or 3LL cancer cells. The antibody treated groups did not show improved antitumor effect compared with vaccine control (p&amp;gt;0.5). To test whether the frequency of memory T-cells recruited to the re-challenged tumor could affect memory T-cell response, antigen-specific pmel-1 T-cells were infused to mice following vaccination with αCTLA-4 or αPD-1. Our result suggested that there were more tumor-infiltrating pmel-1 T-cells in the αCTLA-4 treated group compared to the αPD-1 treated group (p&amp;lt;0.05). In order to augment the durability of αPD-1 treatment, αPD-1 was combined with αCTLA-4 following vaccination. The combined treatment group has superior antitumor response compared to that with αPD-1 (p&amp;lt;0.000003) and overlapped with the αCTLA-4 treated group (P&amp;gt;0.05) during re-challenge, indicating that the effect of the combined treatment is dominated by αCTLA-4. Collectively, our studies facilitate the design of combination immunotherapy treatments that enhance both response rates and generation of memory T-cells to prevent relapse. Citation Format: Stephen Mok, Colm R. Duffy, James P. Allison. Effects of anti-ctla-4 and anti-pd-1 on memory T-cell differentiation and resistance to tumor relapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1685. doi:10.1158/1538-7445.AM2017-1685

Induced Pluripotent Stem Cell-conditioned Medium Suppressed Melanoma Tumorigenicity Through the Enhancement of Natural-Killer Cellular Immunity.

Induced pluripotent stem cells (iPSCs) can secrete cytokines that are involved in T-cell development and affect cytotoxic activity. To assess the effect of iPSC-conditioned medium on tumorigenicity, we retrieved splenocytes from B6 mice and cocultured them with or without irradiated B16 melanoma cells, mouse interleukin-2 (mIL-2), or iPSC-conditioned medium. Splenocyte cytotoxicity assays against B16 melanoma cells [as cytotoxic T lymphocyte (CTL) activity] and P815 cells [as natural killer (NK) activity] were performed. IL-10 and interferon-γ concentrations were measured. An in vivo subcutaneous B16 melanoma growth model was performed in B6 mice and treated with iPSC-conditioned medium. The lymphocyte subpopulation depletion test was performed to determine effectors against B16 melanoma cells. We found that unstimulated splenocytes had little cytotoxic activity. Without tumor cells, mIL-2 could augment iPSC-conditioned medium-treated CTL and NK activities (P<0.01). With irradiated tumor cells, mIL-2 treatment of splenocytes could not enhance CTL or NK activity, but iPSC-conditioned medium could enhance CTL and NK activity (P<0.001). Irradiated tumor cells induced mice splenocytes to secrete more IL-10, similar to mIL-2 treatment, but not iPSC-conditioned medium treatment. mIL-2 had better efficacy than conditioned medium in inducing splenocyte interferon-γ production. The CTL and NK cell depletion test showed that the immunostimulating effect of iPSC-conditioned medium on splenocytes was through the enhancement of NK cellular activity (P<0.05). The subcutaneous melanoma growth model showed that B16-bearing mice treated with an iPSC-conditioned medium intraperitoneal injection had a decreased tumor growth rate (P<0.01). Our study suggests that iPSC-conditioned medium had a protective effect against tumor-induced immunosuppression through the enhancement of host NK cellular activity.

Abstract A038: The feasibility of inducing effective systemic immune responses to human metastatic melanoma with intralesional secreted cytokines

Abstract The induction of new additional immune responses to potential neoantigens on metastatic melanoma resulting in dense lymphocytic infiltrates might cause regression of metastases and could enhance the effects of anti-check point therapies. The blocking of CTLA-4 or PD-1/PD-L1, which normally constrain immune responses, results in significant prolongation of life and up to 7% complete responses (CRs) of metastatic melanoma when used individually. Insufficient nascent anti-melanoma immunity may account for the relatively low frequency of CRs. Also the lack of specificity in the dis-inhibition of immune responses can cause adverse reactions. We investigated a different strategy: inducing new specific immune responses to metastatic melanoma with mixed secreted cytokines. The development of immune responses is regulated by multiple cytokines/chemokines (CKs) acting in concert. To mimic this secretory environment, in a non-randomized prospective study, 88 patients with multiple palpable cutaneous/subcutaneous metastases (stages IIIC and IV-M1a) received weekly injections of autologous secreted CKs into some metastatic nodules while other metastases were never injected. CKs were prepared ex-vivo by stimulating PBMCs with recall microbial antigens to which the patients were sensitive as assessed by PBMC proliferative responses. The secreted mixtures contained individual cytokines at picogram to nanogram concentrations. New immune responses were identified by the development of dense lymphocytic infiltrates in never-injected metastases, consisting of CD8 and smaller numbers of CD4 T cells. This study was conducted before the availability of current therapies. After multiple injections into some metastatic nodules, never-injected metastases developed dense tumor-infiltrating lymphocytes (TILs) and often regressed completely. CD8 TILs from metastases directly killed fresh autologous melanoma cells ex-vivo in an MHC-restricted manner without in- vitro boosting. Although both never-injected and injected metastases often regressed completely, in some patients new metastases appeared even while never- injected nodules were regressing. The new metastases often regressed with or without injections. In 40% of subjects all never-injected and injected metastases regressed completely for &amp;gt; 2 months. 20% remained free of disease for 5 to 28 years (median 60 months, range 2 to 334 months). Median overall survival of patients, progressing and regressing, was 28.5 months (4-348 months), with 55% of patients surviving 2 years, and 30% 5 years. Durable disease-free survival also occurred in recipients of intracutaneous CKs mixed with irradiated autologous melanoma cells. No adverse events occurred. We conclude that intralesional secreted, mixed CKs can induce new systemic and effective cellular immune responses to metastatic melanoma. Mixtures of secreted cytokine/chemokines (CKs) may be more effective in inducing the development of immune responses than individual CKs. These immune responses were associated with a surprisingly high frequency (40%) of complete regressions and prolonged disease-free periods of 1 to 28 years. These new anti-melanoma cellular immune responses also might enhance the frequency of clinical responses to subsequent anti-check point therapies. Citation Format: Fred T. Valentine, Frederick M. Golomb, Matthew Haris, Daniel F. Roses. The feasibility of inducing effective systemic immune responses to human metastatic melanoma with intralesional secreted cytokines. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A038.

T-Cell Responses to Tyrosinase-Derived Self-Peptides in Patients with Leukoderma Induced by Rhododendrol: Implications for Immunotherapy Targeting Melanoma

Background: Rhododendrol, a phenolic compound contained in lightening/whitening cosmetics, can bind and inhibit tyrosinase and was reported to induce leukoderma in Japan. Only 2% of the cosmetics users are affected, and tacrolimus is effective in treatment of the condition. Objective: To test the hypothesis that the disease is an autoimmune disorder. Methods: Short-term T-cell lines were established using peripheral blood mononuclear cells from 8 patients with human melanoma-associated and tyrosinase-derived synthetic peptides. The effects of rhododendrol on melanoma immunization were also examined. Results: Seven out of 8 patients were positive for HLA-DR4. Both class I- and class II-restricted and tyrosinase peptide-specific T-cell responses were observed. Immunization of mice with rhododendrol-treated and irradiated B16 melanoma cells successfully delayed the growth of melanoma cells in vivo. Conclusion: Rhododendrol-induced leukoderma is an autoimmune disorder, with rhododendrol as an environmental factor and HLA-DR4 as a genetic factor. Rhododendrol might be effective in treating melanomas.

Abstract A37: Immunization of cutaneous melanoma patients with the allogeneic cell vaccine CSF-470 enhances immune infiltration of metastatic lesions and would favor subsequent response to Vemurafenib

Abstract Therapeutic cancer vaccines are aimed at promoting tumor-specific immunity with long-term memory. We have developed the CSF-470 therapeutic vaccine, a mini-allograft of four irradiated allogeneic cutaneous melanoma cell lines, combined with BCG and rhGM-CSF as adjuvants; that is currently being assayed in adjuvancy against medium-dose IFN-alfa2b in the CASVAC-0401 phase II/III clinical trial (ClinicalTrials.gov identifier: NCT01729663). An update to September 2014 reveals that after inclusion of 31 patients, a significant benefit in the distant metastasis-free survival for the CSF-470 arm is obtained (p=0.034). Of the 20 patients assigned to the CSF-470 arm, with a mean follow-up of 26 months, six patients developed distant metastases, of whom two died, and fourteen patients are distant metastases-free (70%). Five patients of the vaccine arm were treated with Vemurafenib after progression; two of them achieved complete remission and two of them partial remission (&amp;gt;50%); only one developed severe dermatitis. Before Vemurafenib treatment, metastases biopsies from only three patients could be obtained; two of them later achieved complete responses. In this work, we present the analysis of the peripheral blood and in situ immune populations during CSF-470 immunization in the two patients that achieved complete responses with Vemurafenib treatment. To attain this task, histopathological characterization of tumor biopsies, along with immune populations determined by immunohistochemistry, was performed by microscopy and image analysis. Peripheral blood mononucleated cells were obtained from patients at different times during CSF-470 immunization and immune populations were assessed by flow cytometry. Patient #006 entered the CASVAC-0401 study after a primary and lymph node tumor resection. By the end of CSF-470 immunization, she developed simultaneously subcutaneous and lung metastases. Following a 3-month cycle of Vemurafenib 960mg bid, patient #006 achieved complete remission of lung tumor nodules. Analysis of immune response evolution during protocol revealed a marked increase in in situ immune infiltration at the cutaneous metastasis relative to the primary tumor, especially in CD8+, CD4+ and CD20+ cells. Several dying tumor cells could be distinguished in the cutaneous metastasis, with attached and surrounding CD8+, CD4+ and CD45Ro+ T lymphocytes and CD68+ cells; and with Granzyme B vesicles and DNA strand breaks. Patient #005 developed sequential in transit metastases; CD8+, CD4+ and CD20+ lymphocyte infiltration increased in situ in biopsies following CSF-470 immunization. Vemurafenib treatment allowed achieving a complete response characterized by brisk infiltration of CD68+ macrophages, CD11c+ dendritic cells, CD8+ and CD4+ lymphocytes. In both patients, analysis of peripheral immune populations revealed an increment of NK cells detected after 6-months treatment, followed by an increase in CD4+ and CD8+ cells by the end of the protocol (2 years). Also, serum reactivity to CSF-470 cutaneous melanoma cells increased during vaccination. We suggest that previous immunization of melanoma patients with CSF-470 vaccine increases tumor immune infiltration and subsequent response to Vemurafenib. These encouraging results may open new options for combined therapies in cutaneous melanoma. Citation Format: Mariana Aris, María Betina Pampena, Estrella M. Levy, Alicia I. Bravo, Florencia P. Madorsky-Rowdo, Ana Mordoh, Julio Kaplan, Antonela Baron, Mariela Urrutia, Paula A. Blanco, María Marcela Barrio, José Mordoh. Immunization of cutaneous melanoma patients with the allogeneic cell vaccine CSF-470 enhances immune infiltration of metastatic lesions and would favor subsequent response to Vemurafenib. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A37.