B16 Tumor Growth Research Articles

Abstract 3610: Utilizing fructose metabolism to fuel anti-tumor immunity

Abstract Consumption of fructose in the form of high fructose corn syrup is elevated in the western diet, but it is not fully understood how fructose metabolism influences anti-tumor immunity. Fructose is canonically metabolized in the intestine and the liver and can shunt into glycolysis. In certain tumor types, fructose metabolism becomes deregulated, allowing tumor cells to metabolize fructose to power their biosynthetic processes. Similarly, reprogramming immune cells to be able to efficiently metabolize fructose may help us power immunotherapy to work in difficult to treat cancers, especially in individuals who have high fructose in their blood or in the tumor microenvironment (TME). Imparting effector T cells with the ability to use fructose for fuel can overcome one of the major metabolic limitations of the TME: limited nutrient supply. As T cells become activated, they alter their metabolism to support growth and proliferation. Activated CD8+ T cells upregulate their glycolysis which results in increased biosynthetic processes, such as synthesis of macromolecules and effector cytokines. However, tumor cells also consume substantial amounts of glucose, resulting in a glucose-depleted microenvironment, which could be one of the major barriers to immunotherapy working in solid tumors. Therefore, we engineered T cells to overexpress a fructose transporter, GLUT5, which is normally not expressed in the immune compartment at high levels. We hypothesized that increased fructose uptake by CD8+ T cells will ameliorate exhaustion caused by the glucose-low tumor microenvironment. Indeed, we determined that GLUT5 T cells are more efficient in killing in low glucose supplemented with fructose. We also observed that GLUT5 expression restores T cell glycolytic capacity under fructose to that of unexhausted T cells under glucose replete conditions. Similarly, GLUT5-expressing T cells proliferate faster in vitro and exhibit an improved ability to control B16 tumor growth in vivo. Additionally, combining GLUT5 cell infusion with checkpoint inhibition resulted in durable tumor control and prolonged mouse survival. Further, engineered CD19-targeted CAR T cells with expression of GLUT5 were more metabolically active and cytotoxic in low glucose conditions, mimicking the TME. In conclusion, GLUT5 expression in effector CD8+ and CAR T cells may allow immunotherapy to function in solid tumors under low glucose conditions. Citation Format: Tanya Schild, Rachel Chirayil, Lyric Haughton, Patrick Wallisch, Marjan Berishaj, David A. Scheinberg, Kayvan R. Keshari. Utilizing fructose metabolism to fuel anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3610.

Abstract 4053: Targeting oxygen consumption with metformin and phenformin have differential effects on immune cells in the tumor microenvironment

Abstract T cell immune checkpoint blockade (ICB) has shown remarkable promise in melanoma and other cancers. However, most patients do not show clinical benefit. This is because tumors can activate multiple checkpoints and immunosuppressive pathways to evade anti-tumor immune responses. Inhibition of these immune suppressive mechanisms and immune checkpoints or repolarizing the tumor microenvironment (TME) to become more accessible to the immune system may be necessary for maximal therapeutic efficacy of immunotherapies. There is increasing evidence that tumor hypoxia can attenuate the antitumor immune response by promoting immune suppression and inhibiting direct killing by cytotoxic immune cells. We propose that blocking tumor oxygen consumption using drugs that target the mitochondrial complex I (phenformin and metformin) will enhance the efficacy of immunotherapies such as ICB in preclinical models such as B16 melanoma. In this study, we examined how low oxygen levels (hypoxia) influence T cell effector function such as cytotoxic activity and cytokine production in vitro. We co-cultured B16 melanoma cells in the presence of tumor specific T cells (Pmels) and incubated at 37ºC in either normoxia (~21% O2) or hypoxia (3% O2) and assessed killing and intracellular cytokine production 24 - 48 hours later. We show that the ability of tumor antigen-specific T cells to kill tumor cells or make effector cytokine such as IFNγ and TNFα is significantly reduced in hypoxia settings. We also examined the effects of hypoxia targeting drugs (metformin and phenformin) on T cell priming and activation. When T cells were activated in the presence of each drug in vitro, there was enhanced expression of T cell activation markers (CD25, Granzyme B) as well as an increase in their differentiation into central memory T cells. We next determined whether these drugs could alter the TME in vivo by treating C56BL/6J mice bearing established B16 tumors with phenformin and metformin and then examine the effects on the immune system in the tumor, tumor draining lymph nodes and spleen by flow cytometry. We found that these three drugs have differential effects on both the innate and adaptive the immune system in the tumors and periphery. Finally, we tested whether these drugs could delay B16 melanoma growth in vivo as a monotherapy or in combination with anti-PD1. We found that while both drugs can significantly delay tumor growth as monotherapies, only metformin showed improved anti-tumor efficacy when combined with anti-PD-1 in vivo. These studies show that while both drugs target tumor oxygen consumption (via inhibition of complex I), and can delay B16 tumor growth in vivo, they have differential effects on the TME and will need further considerations when combining with immune based therapies such as ICBs. Citation Format: Sadna Budhu, Anais Assouvie, Mamadou Bah, Svena Verma, Inna Serganova, Mayuresh Mane, Juan Zurita, Jason Koutcher, Vladimir Ponomarev, Jedd D. Wolchok, Taha Merghoub. Targeting oxygen consumption with metformin and phenformin have differential effects on immune cells in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4053.

Opaganib (ABC294640) Induces Immunogenic Tumor Cell Death and Enhances Checkpoint Antibody Therapy.

Antibody-based cancer drugs that target the checkpoint proteins CTLA-4, PD-1 and PD-L1 provide marked improvement in some patients with deadly diseases such as lung cancer and melanoma. However, most patients are either unresponsive or relapse following an initial response, underscoring the need for further improvement in immunotherapy. Certain drugs induce immunogenic cell death (ICD) in tumor cells in which the dying cells promote immunologic responses in the host that may enhance the in vivo activity of checkpoint antibodies. Sphingolipid metabolism is a key pathway in cancer biology, in which ceramides and sphingosine 1-phosphate (S1P) regulate tumor cell death, proliferation and drug resistance, as well as host inflammation and immunity. In particular, sphingosine kinases are key sites for manipulation of the ceramide/S1P balance that regulates tumor cell proliferation and sensitivity to radiation and chemotherapy. We and others have demonstrated that inhibition of sphingosine kinase-2 by the small-molecule investigational drug opaganib (formerly ABC294640) kills tumor cells and increases their sensitivities to other drugs and radiation. Because sphingolipids have been shown to regulate ICD, opaganib may induce ICD and improve the efficacy of checkpoint antibodies for cancer therapy. This was demonstrated by showing that in vitro treatment with opaganib increases the surface expression of the ICD marker calreticulin on a variety of tumor cell types. In vivo confirmation was achieved using the gold standard immunization assay in which B16 melanoma, Lewis lung carcinoma (LLC) or Neuro-2a neuroblastoma cells were treated with opaganib in vitro and then injected subcutaneously into syngeneic mice, followed by implantation of untreated tumor cells 7 days later. In all cases, immunization with opaganib-treated cells strongly suppressed the growth of subsequently injected tumor cells. Interestingly, opaganib treatment induced crossover immunity in that opaganib-treated B16 cells suppressed the growth of both untreated B16 and LLC cells and opaganib-treated LLC cells inhibited the growth of both untreated LLC and B16 cells. Next, the effects of opaganib in combination with a checkpoint antibody on tumor growth in vivo were assessed. Opaganib and anti-PD-1 antibody each slowed the growth of B16 tumors and improved mouse survival, while the combination of opaganib plus anti-PD-1 strongly suppressed tumor growth and improved survival (p < 0.0001). Individually, opaganib and anti-CTLA-4 antibody had modest effects on the growth of LLC tumors and mouse survival, whereas the combination of opaganib with anti-CTLA-4 substantially inhibited tumor growth and increased survival (p < 0.001). Finally, the survival of mice bearing B16 tumors was only marginally improved by opaganib or anti-PD-L1 antibody alone but was nearly doubled by the drugs in combination (p < 0.005). Overall, these studies demonstrate the ability of opaganib to induce ICD in tumor cells, which improves the antitumor activity of checkpoint antibodies.

IL-21 or synthetic IL-2 treatment drives T cell IFNγ production or expansion, respectively, during adoptive cell therapy of melanoma

Abstract Adoptive cell therapy (ACT) of tumor-infiltrating lymphocytes (TILs) can generate long-term durable responses in patients with melanoma and can even successfully treat patients that are refractory to anti-PD-1 checkpoint blockade. IL-2 is often used to expand TILs, however the administration of this cytokine can induce systemic toxicities in patients. Synthetic cytokine mimetics of IL-2 have been developed, including neoleukin-2 (Neo2), which has been shown to have enhanced therapeutic efficacy over IL-2, with reduced toxicity. IL-21 has also been used in TIL therapy to promote T cell expansion. Here, we tested the efficacy of Neo2 or IL-21 in treating mice bearing B16 tumors that received ACT with melanoma-specific T cells (TRP1 Highand TRP1 Low; TCRs recognizing antigen with high or low affinity). Both Neo2 and IL-21 could slow growth of B16 tumors, curing several mice. We performed single-cell RNA-sequencing on these tumors to analyze cytokine therapy-induced changes in the microenvironment. Neo2-treated tumors had a signature primarily of T cell expansion, with few other associated transcriptional changes. IL-21 therapy generated a profound IFNγ signature, with an increase in T cells production of IFNγ and many cell types, including T cells, macrophages, and tumor cells responding with interferon-stimulated gene expression. Interestingly, IL-21 therapy did not induce profound T cell expansion as Neo2 did, but importantly both IL-21 and Neo2 therapy reduced levels of Tregs within the tumor. IL-21 also improved T cell production of granzyme B. Thus, IL-2 primarily expands T cells and IL-21 generates higher quality anti-tumor T cells. Therefore, IL-21 is a promising cytokine for improving ACT and other immunotherapies.

Supramolecular assembly of a trivalent peptide hydrogel vaccine for cancer immunotherapy.

Vaccination shows great promise in cancer immunotherapy. However, the induction of robust and broad therapeutic CD8 T cell immunity against tumors is challenging due to the essential heterogenicity of tumor antigen expression. Recently, bioinspired materials have reshaped the field of cancer nanomedicine. Herein, a bioinspired nanofibrous trivalent peptide hydrogel vaccine was constructed using the spontaneous supramolecular co-assembly of three antigenic epitope-conjugated peptides, which could mimic the fibrillar structure and biological function of the extracellular matrix and naturally occurring protein assembly. The hydrogel vaccine could be accurately and flexibly adjusted to load each antigenic peptide at a defined ratio, which facilitated the antigen presentation of dendritic cells and significantly improved the initiation of CD8 T cell response and the secretion of interferon-γ (IFN-γ). C57BL/6 mice were immunized with the trivalent peptide hydrogel vaccine, where it elicited a high broad-spectrum antitumor CD8 T cell response that significantly inhibited the growth of B16 tumors in the absence of additional immunoadjuvants or delivery systems. In summary, the supramolecular assembly of triple antigenic epitope-conjugated peptides offers a simple, customizable, and versatile approach for the development of cancer vaccines with remarkable therapeutic efficacy, thereby providing a highly versatile platform for the application of personalized multivalent tumor vaccines. STATEMENT OF SIGNIFICANCE: (1) We report a feasible, versatile and bioinspired approach to manufacture a multivalent peptide-based hydrogel cancer vaccine in the absence of additional adjuvants, which closely mimics immune niches, co-delivers antigen epitopes, greatly promotes antigen presentation to DCs and their subsequent homing to dLNs and elicits a broad-spectrum antitumor CD8 T cell response, resulting in significant inhibition of B16 tumor growth. (2) This feasible and efficient co-assembly strategy provides an attractive platform for engineering a range of multivalent vaccines at defined ratios to further enhance antigen-specific T cell responses. This approach may also be used for personalized immunotherapy with neo-epitopes.

Pre-treatment of oncolytic reovirus improves tumor accumulation and intratumoral distribution of PEG-liposomes.

PEGylated liposomes (PEG-liposomes) are a promising drug delivery vehicle for tumor targeting because of their efficient tumor disposition profiles via the enhanced permeability and retention (EPR) effect. However, tumor targeting of PEG-liposomes, particularly their delivery inside the tumors, is often disturbed by physical barriers in the tumor, including tumor cells themselves, extracellular matrices, and interstitial pressures. In this study, B16 melanoma tumor-bearing mice were injected intravenously with oncolytic reovirus before administration of PEG-liposomes to enhance PEG-liposomes' tumor disposition. Three days after reovirus administration, significant expression of reovirus sigma 3 protein, elevation of apoptosis-related gene expression, and activation of caspase 3 in the tumors were found. Apoptotic cells were found inside the tumors. These data indicated that reovirus efficiently replicated in the tumors and induced apoptosis of tumor cells. The tumor disposition levels of PEG-liposomes were approximately doubled by reovirus pre-administration, compared with a PBS-pretreated group. PEG-liposomes were widely distributed in the tumors of reovirus-pretreated mice, whereas in the PBS-pretreated group, PEG-liposomes were found mainly around or inside the blood vessels in the tumors. Pre-treatment with reovirus also improved the tumor accumulation of PEG-liposomes in human pancreatic BxPC-3 tumors. 3D imaging analysis of whole BxPC-3 tumors demonstrated that pretreatment with reovirus led to the enhancement of PEG-liposome accumulation inside the tumors. Combination treatment with reovirus and paclitaxel-loaded PEG-liposomes (PTX-PEG-liposomes) significantly suppressed B16 tumor growth. These results provide important information for clinical use of combination therapy of reovirus and nanoparticle-based drug delivery system (DDS).

Comparative Analysis of the Pharmacological Activity of Bis(3,5-di-Tert-Butyl-4-Hydroxyphenylthiolate)Dimethylol in Different Modes of Administration on a Mouse Model of Melanoma B16 Tumor Growth.

We performed a comparative analysis of the pharmacological activity of the hybrid organotin compound bis(3,5-di-tert-butyl-4-hydroxyphenylthiolate)dimethylol (Me-3) administered in different modes to mice with transplanted melanoma B16 to identify the most effective dosage regimen. Three modes of administration were used: preventive (before transplantation of tumor cells), classical according to Z. P. Sof'ina (48 h after transplantation of tumor cells), and delayed (7 days after transplantation of tumor cells, after formation of nodules of the primary tumor node). Compound Me-3 was administered at a total dose of 375 mg/kg intraperitoneally once a day for 5 days. The classical mode of administration was identified as the most effective, which indicates the preventive antimetastatic activity of Me-3 on the model of the transplanted mouse tumor melanoma B16.

PD-1 inhibition on pulmonary ILC2s promotes TNF-α production and restricts progression of metastatic melanoma tumor growth

Abstract Pulmonary metastatic melanoma is an aggressive form of cancer that is highly resistant to treatment and research efforts targeting this cancer are severely lacking. While pulmonary Group 2 Innate lymphoid cells (ILC2s) represent one of the major tissue-resident innate lymphoid cell populations at steady state and are key drivers of cytokine secretion in their occupational niche, their role in pulmonary cancer progression remains unclear. As the programmed cell death protein-1 (PD-1) plays a major role in cancer immunotherapy and immunoregulatory properties, here we investigated the specific effect of PD-1 inhibition on ILC2s during a pulmonary murine model of melanoma cancer metastasis. We demonstrate that murine models of B16 tumor growth upregulate PD-1 expression on pulmonary ILC2s and that this expression severely promotes tumor growth. Conversely, utilizing targeted murine adoptive transfers we demonstrate that PD-1 inhibition on ILC2s specifically and significantly inhibits tumor growth. Furthermore, the results revealed that PD-1 inhibition upregulated both murine and human ILC2 TNF-α production and secretion, a cytotoxic cytokine that we demonstrate has the ability to directly induce tumor cell death and apoptosis independent of adaptive immunity. Together, the results of these studies may lead to a better understanding of ILC2s and their role in metastatic melanoma, as well as providing a foundation for further elucidation of their anti-tumor mechanisms in cancer progression. Supported by grants from NIH (R01 ES025786, R01 ES021801, R01 HL144790, R01 HL151493, R01 AI145813 and R01 HL151769)

Targeting Endothelial Connexin37 Reduces Angiogenesis and Decreases Tumor Growth.

Connexin37 (Cx37) and Cx40 form intercellular channels between endothelial cells (EC), which contribute to the regulation of the functions of vessels. We previously documented the participation of both Cx in developmental angiogenesis and have further shown that loss of Cx40 decreases the growth of different tumors. Here, we report that loss of Cx37 reduces (1) the in vitro proliferation of primary human EC; (2) the vascularization of subcutaneously implanted matrigel plugs in Cx37−/− mice or in WT using matrigel plugs supplemented with a peptide targeting Cx37 channels; (3) tumor angiogenesis; and (4) the growth of TC-1 and B16 tumors, resulting in a longer mice survival. We further document that Cx37 and Cx40 function in a collaborative manner to promote tumor growth, inasmuch as the injection of a peptide targeting Cx40 into Cx37−/− mice decreased the growth of TC-1 tumors to a larger extent than after loss of Cx37. This loss did not alter vessel perfusion, mural cells coverage and tumor hypoxia compared to tumors grown in WT mice. The data show that Cx37 is relevant for the control of EC proliferation and growth in different tumor models, suggesting that it may be a target, alone or in combination with Cx40, in the development of anti-tumoral treatments.

PD-1 Blockade on Tumor Microenvironment-Resident ILC2s Promotes TNF-α Production and Restricts Progression of Metastatic Melanoma.

While pulmonary ILC2s represent one of the major tissue-resident innate lymphoid cell populations at steady state and are key drivers of cytokine secretion in their occupational niche, their role in pulmonary cancer progression remains unclear. As the programmed cell death protein-1 (PD-1) plays a major role in cancer immunotherapy and immunoregulatory properties, here we investigate the specific effect of PD-1 inhibition on ILC2s during pulmonary B16 melanoma cancer metastasis. We demonstrate that PD-1 inhibition on ILC2s suppresses B16 tumor growth. Further, PD-1 inhibition upregulates pulmonary ILC2-derived TNF-α production, a cytotoxic cytokine that directly induces cell death in B16 cells, independent of adaptive immunity. Together, these results highlight the importance of ILC2s and their anti-tumor role in pulmonary B16 cancer progression during PD-1 inhibitory immunotherapy.

Treatment of Melanoma by Nano-conjugate-Delivered Wee1 siRNA.

Small interfering RNA (siRNA)-based drugs have shown tremendous potential to date in cancer gene therapy. Despite the considerable efforts in siRNA design and manufacturing, unsatisfactory delivery systems persist as a limitation for the application of siRNA-based drugs. In this work, the cholesterol, cell-penetrating peptide conjugate cRGD (R8-cRGD), and polyethylene glycol (PEG) were introduced into low-molecular-weight polyethyleneimine (LMW PEI) to form cRGD-R9-cholesterol-PEI-PEG (RRCPP) nanoparticles with specific targeting and highly penetrating abilities. The enhanced siRNA uptake efficiency of the RRCPP delivery system benefited from R8-cRGD modification. Wee1 is an oncogenic nuclear kinase that can regulate the cell cycle as a crucial G2/M checkpoint. Overexpression of Wee1 in melanoma may lead to a poor prognosis. In the present study, RRCPP nanoparticles were designed for Wee1 siRNA delivery to form an RRCPP/siWee1 complex, which significantly silenced the expression of the WEE1 gene (>60% inhibition) and induced B16 tumor cell apoptosis by abrogating the G2M checkpoint and DNA damage in vitro. Furthermore, the RRCPP/siWee1 complex suppressed B16 tumor growth in a subcutaneous xenograft model (nearly 85% inhibition rate) and lung metastasis (nearly 66% inhibition rate) with ideal in vivo safety. Briefly, our results support the validity of RRCPP as a potential Wee1 siRNA carrier for melanoma gene therapy.

Abstract NG12: From cholesterol regulation to tumor suppression: Pcsk9 as a novel target for cancer immunotherapy

Abstract NG12: From cholesterol regulation to tumor suppression: Pcsk9 as a novel target for cancer immunotherapy

A novel cancer immunotherapy using tumor-infiltrating B cells in the APCmin/+ mouse model.

Accumulating evidence has suggested a correlation of tumor infiltrating B cells (TiBcs) and a good prognosis of cancer diseases. In some cases, TiBcs appear to have experienced antigen stimulation since they have undergone class-switching and somatic hypermutation and formed tertiary lymphoid structures around tumors together with T cells. Assuming TiBcs include those that recognize some tumor antigens, we sought to investigate their possible usefulness for cell-mediated immunotherapies. To expand usually small number of TiBcs in vitro, we modified our B cell culture system: we transduced B cells with ERT2-Bach2 so that they grow unlimitedly provided with tamoxifen, IL-21 and our original feeder cells. Such cells differentiate into plasma cells and produce antibodies upon withdrawal of tamoxifen, and further by addition of a Bach2-inhibitor in vitro. As a preliminary experiment, thus expanded splenic B cells expressing a transgenic antigen receptor/antibody against hen egg lysozyme were intravenously injected into mice pre-implanted with B16 melanoma cells expressing membrane-bound HEL in the skin, which resulted in suppression of the growth of B16 tumors and prolonged survival of the recipient mice. To test the usefulness of TiBcs for the immunotherapy, we next used APCmin/+ mice as a model that spontaneously develop intestinal tumors. We cultured TiBcs separated from the tumors of APCmin/+ mice as above and confirmed that the antibodies they produce recognize the APCmin/+ tumor. Repeated injection of such TiBcs into adult APCmin/+ mice resulted in suppression of intestinal tumor growth and elongation of the survival of the recipient mice. Serum antibody from the TiBc-recipient mice selectively bound to an antigen expressed in the tumor of APCmin/+ mice. These data suggest a possibility of the novel individualized cancer immunotherapy, in which TiBcs from surgically excised tumor tissues are expanded and infused into the donor patients.

Steatohepatitis Impairs T-cell–Directed Immunotherapies Against Liver Tumors in Mice

Nonalcoholic steatohepatitis causes loss of hepatic CD4+ T cells and promotes tumor growth. The liver is the most common site of distant metastases from a variety of malignancies, many of which respond to immunotherapy. We investigated the effects of steatohepatitis on the efficacy of immunotherapeutic agents against liver tumors in mice. Steatohepatitis was induced by feeding C57BL/6NCrl or BALB/c AnNCr mice a methionine and choline-deficient diet or a choline-deficient l-amino acid-defined diet. Mice were given intrahepatic or subcutaneous injections of B16 melanoma and CT26 colon cancer cells, followed by intravenous injections of M30-RNA vaccine (M30) or intraperitoneal injections of an antibody against OX40 (aOX40) on days 3, 7, and 10 after injection of the tumor cells. We measured tumor growth and analyzed immune cells in tumor tissues by flow cytometry. Mice were given N-acetylcysteine to prevent loss of CD4+ T cells from liver. Administration of M30 and aOX40 inhibited growth of tumors from intrahepatic injections of B16 or CT26 cells in mice on regular diet. However, M30 and/or aOX40 did not slow growth of liver tumors from B16 or CT26 cells in mice with diet-induced steatohepatitis (methionine and choline-deficient diet or choline-deficient l-amino acid-defined diet). Steatohepatitis did not affect the ability of M30 to slow growth of subcutaneous B16 tumors. In mice with steatohepatitis given N-acetylcysteine, which prevents loss of CD4+ T cells, M30 and aOX40 were able slow growth of hepatic tumors. Flow cytometry analysis of liver tumors revealed reduced CD4+ T cells and effector memory cells in mice with vs without steatohepatitis. Steatohepatitis reduces the abilities of immunotherapeutic agents, such as M30 and aOX40, to inhibit tumor liver growth by reducing tumor infiltration by CD4+ T cells and effector memory cells. N-acetylcysteine restores T-cell numbers in tumors and increases the ability of M30 and aOX40 to slow tumor growth in mice.

A chemical conjugation of JQ-1 and a TLR7 agonist induces tumoricidal effects in a murine model of melanoma via enhanced immunomodulation.

In recent years, inhibitors of the BET bromodomain proteins, such as BRD4 inhibitors, have demonstrated robust antitumor activity. JQ-1, a representative small molecular BRD4 inhibitor, is also effective to block PD-1/PD-L1 signaling by significantly decreasing the PD-L1 expression on tumor cells. However, toxicity of BRD4 inhibitors on lymphoid and hematopoietic tissues limits their clinical usage. In this research, we designed and studied an immunogenic BRD4 inhibitor, SZU-119, by coupling JQ-1 with a TLR7 agonist, SZU-101. In vitro, SZU-119 stimulated the production of cytokines in mouse BMDCs and spleen lymphocytes, and inhibited the expression of PD-L1 in mouse B16 tumor cells. In vivo, SZU-119 suppressed the B16 tumor growth at both injected and uninjected sites, and prolonged the survival time of mice. SZU-119 elevated the number of total CD8+ and IFN-γ+ CD8+ T cells in spleens, with greater CTL cytotoxicity to B16 tumor cells. It was also observed that the infiltration of CD8+ T cells was increased in tumors at both local and distant sites, and the PD-L1 expression was decreased in tumor cells at the primary site. In conclusion, we have demonstrated that SZU-119 activated the innate immune cells, kept efficacy of PD-L1 blockade and abrogated immune toxicity, showing more potent antitumor effects than the simple mixture of SZU-101 and JQ-1 in a mouse melanoma model. Our work provides new insights for the development of anti-melanoma drugs that concurrently target innate and adaptive immunity.

A recombinant oncolytic Newcastle virus expressing MIP-3α promotes systemic antitumor immunity

BackgroundThe oncolytic Newcastle disease virus (NDV) is inherently able to trigger the lysis of tumor cells and induce the immunogenic cell death (ICD) of tumor cells and is also an...

A traditional Chinese medicine formula inhibits tumor growth in mice and regulates the miR-34b/c-Met/β-catenin pathway

Ethnopharmacology relevanceSi-Jun-Zi-Tang (SJZT) is a traditional Chinese medicine formula used to treat chronic and debilitating diseases including melanoma. SJZT-based therapies have achieved good clinical outcomes in melanoma management. However, the pharmacological basis of SJZT for its clinical use in melanoma treatment is not fully understood. Aim of the studyTo investigate the anti-melanoma effects and mechanism of action of an ethanolic extract of SJZT. Materials and methodsSJZT was extracted using 50% ethanol. A murine B16 melanoma-bearing mouse model was employed to investigate the anti-melanoma effects of SJZT. microRNA (miRNA) and mRNA levels were examined by RT-qPCR, and protein levels were measured by Western blotting. ResultsSJZT significantly inhibited B16 tumor growth in mice. Mechanistic investigations revealed that SJZT elevated miR-34b (a tumor suppressing miRNA), and lowered c-Met (a miR-34b target gene) and β-catenin (a downstream molecule of c-Met signaling) expression levels in the B16 tumors. ConclusionsIn this study we found, for the first time, that SJZT exerts anti-melanoma effects and regulates the miR-34b/c-Met/β-catenin pathway in a melanoma mouse model. Our findings provide pharmacological justifications for the clinical use of SJZT in treating melanoma.

Suppressive effects of fucoidan-agaricus mix (CUA-fucoidan) on angiogenesis and tumor growth in B16 melanoma-bearing mice

Abstract Fucoidan is a series of natural sulfated polysaccharides contained in brown sea algae. In previous studies, we proved that fucoidans derived from Cladosiphon okamuranus and Undaria pinnatifida effectively augmented anti-tumor immunity in combination with an Agaricus blazei mycelium extract. In this study, we evaluated dietary effects of the mixture consist of the two high-molecular weights fucoidan and the agaricus extract (named CUA-fucoidan) on tumor angiogenesis. For that purpose, C57BL/6 mice were subcutaneously injected basement membrane matrix (Geltrex) enclosing 5×104 mitomycin C-treated B16 melanoma cells or 500 ng/mL vascular endothelial growth factor (VEGF), and fed 2% CUA-fucoidan containing diet for 7 days before and after the matrix inoculation. The accumulation of hemoglobin and CD31-positive cells in the B16-enclosed Geltrex was lower in the CUA-fucoidan-fed mice than in the control mice, whereas the hemoglobin contents in the VEGF-enclosed Geltrex were not affected by the CUA-fucoidan feeding. These results suggested that CUA-fucoidan was effective in suppressing tumor angiogenesis, but did not inhibit the physiological action of VEGF. Rather, it was guessed that CUA-fucoidan suppressed VEGF production because the expression of VEGF mRNA was reduced in the matrix resected from CUA-fucoidan-fed mice. In this context, the infiltration of CD206-positive macrophages, which known to produce VEGF, were declined by the CUA-fucoidan feeding. Furthermore, CUA-fucoidan decreased accumulation of Treg in the B16-included Geltrex, and delayed B16 tumor growth. In conclusion, CUA-fucoidan was expected to be effective in inhibition of tumor angiogenesis by improving immunosuppressive tumor microenvironments.

Abstract B25: Blockade of Stat3 oncogene addiction induces cellular senescence and reveals a cell-nonautonomous activity suitable for cancer immunotherapy

Abstract Stat3 is constitutively activated in diverse cancers and acts as a critical mediator of tumor immune evasion. Previously, we described in murine breast cancer (BC) models that blockade of Stat3 activation induces cellular senescence. Although senescent cells are growth arrested, they remain metabolically active and develop a senescence-associated secretory phenotype (SASP) that can have pro- as well as antitumorigenic effects. Our objectives were to characterize the composition and activity of the SASP induced by Stat3 blockade (SASP-Stat3) and to develop an immunotherapy (IT) based on this SASP. Here we report that Stat3 knockdown induced senescence and growth arrest only in tumor cells that exhibit constitutive activation of this oncogene (oncogene addiction), such as 4T1 BC and B16-OVA (herein “B16”) melanoma cells. Moreover, we observed that the SASP-Stat3 from 4T1 and B16 cells had several layers of antitumor effects. One of the effects relied on inhibition of tumor cell proliferation, migration and angiogenic activity. The other enhanced T-cell proliferation and activation in vitro. Furthermore, these effects were not observed with the conditioned medium (CM) from Stat3-blocked MCA cells, which are not Stat3 addicted. In order to translate these findings to a potential clinical application, we designed an immunization protocol based on the administration of irradiated wild-type cancer cells together with a depot of the SASP-Stat3. Therapeutic IT with SASP-Stat3 in mice bearing 4T1 or B16 tumors decreased tumor growth compared with control CM (CM-Control). In 4T1 tumors, we also observed a decrease in pulmonary metastasis vs. CM-Control. In addition, combination of the SASP-Stat3-based IT with an anti PD-1 antibody enhanced the antitumor activity against B16 tumor growth. We observed that this synergistic antitumor effect was the result of the involvement of different subsets of immune cells: SASP-Stat3-based IT activates CD4+ T cells and NK cells, while anti PD-1 therapy targets CD8+ T cells. Next, we studied the composition of the SASP-Stat3 through cytokine array and proteomic studies and disclosed several T cells and NK cells-attracting chemokines, IFNγ-induced cytokines and IFN-associated proteins (IP-10, RANTES, IL-15, MCP-1, ISG15 and IFI35). Taken together, these results demonstrate that Stat3 blockade in tumor cells that are addicted to this oncogene results in the induction of cellular senescence with the production of a SASP that has antitumoral and immune-stimulating activities. Cytokines and proteins from the SASP can be used to formulate an effective adjuvant to enhance the antitumor effect of anti-PD-1 antibodies. Citation Format: Mara De Martino, Mercedes Tkach, María F. Mercogliano, Mauro E. Cenciarini, María F. Chervo, Cecilia J. Proietti, Patricia V. Elizalde, Eliane Piaggio, Roxana Schillaci. Blockade of Stat3 oncogene addiction induces cellular senescence and reveals a cell-nonautonomous activity suitable for cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B25.

Mouse Melanoma B16 Tumors are Soft and Engulfable when Targeted in Combination with Macrophage Checkpoint Blockade

Murine B16 melanoma is a widely used preclinical model for evaluating cancer immunotherapies, and its mechanical properties are likely to influence immune cell interactions and their consequences. Pipette aspiration rheology shows B16 tumors are very soft (relative to normal skin or liver), deform irreversibly at stresses some cells can generate (>>1 kPa), and sometimes fragment. The softness of these tumors is consistent with a relative lack of key fibrillar proteins and with a rapid growth rate that likely outpaces matrix deposition by stromal cells. How these physicochemical characteristics of the tumor microenvironment might modulate phagocytosis remains poorly understood, but macrophages have the machinery to generate considerable force and could overcome cell-cell cohesion. Phagocytosis of B16 cells by primary marrow-derived mouse macrophages in vitro is efficient with a combination of tumor-opsonizing antibody against a B16 surface antigen and function-blocking antibodies against the macrophage receptor SIRPα that binds ubiquitously expressed CD47 in a ‘macrophage checkpoint’. Similarly prepared, fresh marrow monocytes are also injected systemically as a cell-based therapy that slows or prevents B16 tumor growth. Importantly, we do not observe anemia, a major clinical side effect of CD47 blocking antibodies caused by clearance of blood cells. Cancer cells that strongly bind to matrix or one another might not be engulfed as readily as softer tumors.