B16F10 Tumor Research Articles

Antioxidant Activities of Commiphora leptophloeos (Mart.) J. B. Gillett) (Burseraceae) Leaf Extracts Using In Vitro and In Vivo Assays.

Commiphora leptophloeos is widely used in folk medicine without any scientific basis. Considering this, the aim of this study was to evaluate the chemical profile and the antioxidant activity of C. leptophloeos leaf extracts using in vitro and in vivo assays. Six extracts were obtained from fresh leaves using a serial extraction (nonpolar to polar solvents). These extracts were first evaluated with the presence of phytochemical compounds using the methods thin layer chromatography (TLC), ultrahigh performance liquid chromatography (UHPLC-DAD), and high performance liquid chromatography, both with diode array detection (HPLC-DAD). Based on the compounds identified, it was used some bioinformatics tools in order to identify possible pathway and gene targets. After that, the antioxidant capacity from these extracts was analysed by in vitro assays and in vivo assays using Caenorhabditis elegans model. Phytochemical analyses showed the presence of polyphenols, such as rutin, vitexin, and quercetin diglycosides in all extracts, especially in ethanol extract (EE) and methanol extract (EM). Bioinformatics analysis showed these polyphenols linked to antioxidant pathways. Furthermore, EE and EM displayed a high antioxidant capacity in DPPH and superoxide radical scavenging assays. They also had no effect on cell viability for 3T3 nontumour cell. However, for B16-F10 tumour cell lines, these extracts had toxicity effect. In vivo assays using C. elegans N2 showed that EE was not toxic, and it did not affect its viability nor its development. Besides, EE increased worm survival under oxidative stress and reduced intracellular reactive oxygen species (ROS) levels by 50%. Thus, C. leptophloeos EE displayed an important in vitro and in vivo antioxidant capacity. The EE extract has polyphenols, suggesting that these compounds may be responsible for a myriad of biological activities having this potential to be used in various biotechnological applications.

Combination immunotherapy of chlorogenic acid liposomes modified with sialic acid and PD-1 blockers effectively enhances the anti-tumor immune response and therapeutic effects

Melanoma is one of the most common malignant tumors. The anti-PD-1 antibody is used for the treatment of metastatic melanoma. Treatment success is only 35–40% and a range of immune-related adverse reactions can occur. Combination of anti-PD1 antibody therapy with other oncology therapies has been attempted. Herein, we assessed whether chlorogenic acid liposomes modified with sialic acid (CA-SAL) combined with anti-PD1 antibody treatment was efficacious as immunotherapy for melanoma. CA-SAL liposomes were prepared and characterized. In a mouse model of B16F10 tumor, mice were treated with an anti-PD1 antibody, CA-SAL, or combination of CA-SAL + anti-PD1 antibody, and compared with no treatment controls. The tumor inhibition rate, tumor-associated macrophages (TAMs) phenotype, T-cell activity, and safety were investigated. We observed a significant decrease in the proportion of M2-TAMs and CD4+Fop3+ T cells, while there was a significant increase in the proportion of M1-TAMs and CD8+ T cells, and in the activity of T cells, and thus in the tumor inhibition rate. No significant toxicity was observed in major organs. CA-SAL and anti-PD1 Ab combination therapy presented synergistic anti-tumor activity, which enhanced the efficacy of the PD-1 checkpoint blocker in a mouse model of melanoma. In summary, combination immunotherapy of CA-SAL and anti-PD1 Ab has broad prospects in improving the therapeutic effect of melanoma, and may provide a new strategy for clinical treatment.

SLAMF7 Signaling Reprograms T Cells toward Exhaustion in the Tumor Microenvironment.

T cell exhaustion represents one of the most pervasive strategies tumors employ to circumvent the immune system. Although repetitive, cognate TCR signaling is recognized as the primary driving force behind this phenomenon, and it remains unknown what other forces drive T cell exhaustion in the tumor microenvironment (TME). In this study, we show that activation of the self-ligand SLAMF7 immune receptor on T cells induced STAT1 and STAT3 phosphorylation, expression of multiple inhibitory receptors, and transcription factors associated with T cell exhaustion. Analysis of The Cancer Genome Atlas revealed that SLAMF7 transcript levels were strongly correlated with various inhibitory receptors and that high SLAMF7 expression was indicative of poor survival in clear cell renal cell carcinoma (ccRCC). Targeted reanalysis of a CyTOF dataset, which profiled the TME in 73 ccRCC patients, revealed cell-type-specific SLAMF7 expression patterns, strong correlations between exhausted T cells and SLAMF7+ tumor-associated macrophages (TAMs), and a unique subset of SLAMF7highCD38high TAMs. These SLAMF7highCD38high TAMs showed the strongest correlations with exhausted T cells and were an independent prognostic factor in ccRCC. Confirmatory ex vivo coculture studies validated that SLAMF7-SLAMF7 interactions between murine TAMs and CD8+ T cells induce expression of multiple inhibitory receptors. Finally, mice lacking SLAMF7 show restricted growth of B16-F10 tumors, and CD8+ T cells from these mice express less PD-1 and TOX and exhibited an impaired ability to progress through the exhaustion developmental trajectory to terminal exhaustion. These findings suggest that SLAMF7 might play an important role in modulating T cell function in the TME.

Self-Adjuvanted Molecular Activator (SeaMac) Nanovaccines Promote Cancer Immunotherapy.

Neoantigen-based immunotherapy is a promising treatment option for many types of cancer. However, its efficacy and abscopal effect are limited by impotent neoantigens, high treatment costs, and complications due to action of adjuvants. Here, the design and synthesis of nanovaccines are reported, based on self-adjuvanted, polymer nanoparticles with in vivo neoantigen-harvesting and molecular activating capabilities. These nanovaccines inhibit tumor growth significantly and prolong the survival of tumor-bearing mice in both colon carcinoma 26 (CT26) and B16-F10 tumor models. Mechanistic studies suggest that as-synthesized nanovaccines can promote dendritic cell maturation and accumulation expeditiously in lymph nodes, leading to the expansion of cytotoxic CD8+ T cells. Moreover, these nanovaccines elicit abscopal effects in CT26 and B16-F10 tumors without the need for adjuvants or immune checkpoint inhibitors. Combined with an anti-PD-L1 antibody, nanovaccines can evoke robust, long-term memory immune response, as evidenced by tumor growth inhibition and high survival rates (∼ 67%) over 90 days. These results highlight the potential of using self-adjuvanted nanovaccines as a simple, safe, and affordable strategy to boost neoantigen-based cancer immunotherapy.

A two-herb formula inhibits STAT3 signaling and exerts anti-melanoma effects in cell and animal models

Ethnopharmacological relevanceMalignant melanoma is a fatal cancer. Signal transducer and activator of transcription 3 (STAT3) has been proposed as a therapeutic target of melanoma. An herbal formula Huai-Hua-San (HHS) comprising Sophorae Flos (SF) and Gardeniae Fructus (GF) is traditionally used for treating cancers including melanoma, but the pharmacological basis is unknown. Aims of this studyThis study aimed to investigate the anti-melanoma effects of an ethanolic extract of HHS (HHSE), and explore the involvement of STAT3 signaling in the effects. Materials and methodsAn UPLC-TOF/MS method was developed to control the quality of HHSE. A B16F10 allograft mouse model and three melanoma cell lines (B16F10, A375 and A2058) were used to determine the anti-melanoma effects of HHSE. Dacarbazine (DTIC) and Stattic were used as positive controls. Cell viability was detected using MTT and crystal violet staining assays. Cell apoptosis was analyzed by flow cytometry after the cells were stained with Annexin-V/PI. Cell invasive ability was examined using the transwell assay. Protein levels were determined by Western blotting. ResultsThe contents of crocin I, crocin II, quercetin and kaempferol in HHSE were 0.59%, 0.98%, 4.66% and 1.15%, respectively. A clinically relevant dose of HHSE (0.1 g/kg/day, i.g. for 15 consecutive days) significantly suppressed B16F10 tumor growth in mice. HHSE dose-dependently reduced cell viability and dampened invasion of, and induced apoptosis in, melanoma cells. Mechanistic studies revealed that HHSE inhibited the phosphorylation/activation of STAT3 in B16F10 allografts and in cultured melanoma cells. In cell models, HHSE also inhibited the phosphorylation of STAT3 upstream kinases, JAK2 (Tyr1007/1008) and Src (Tyr416), lowered STAT3 nuclear levels, and down-regulated the protein levels of STAT3-targeted molecules. Over-activation of STAT3 in A375 cells significantly attenuated the cytotoxic effects of HHSE. ConclusionsHHSE exhibits anti-melanoma effects in cell and mouse models. Inhibition of STAT3 signaling contributes to the anti-melanoma mechanisms of HHSE. Our findings lay a groundwork for developing HHSE as a modern agent for melanoma management, and provide pharmacological justifications for the traditional use of HHS in treating melanoma.

Growth Hormone Upregulates Mediators of Melanoma Drug Efflux and Epithelial-to-Mesenchymal Transition In Vitro and In Vivo.

Simple SummaryGrowth hormone (GH) action is strongly implicated in the progression and therapy resistance in several types of solid tumors which overexpress the GH receptor (GHR). The aim of our study was to characterize the effects of GH and its downstream effector insulin-like growth factor 1 (IGF-1) on melanoma using in vitro and in vivo models. We confirmed an IGF-1-independent role of elevated circulating GH in upregulating key mechanisms of therapy resistance and malignancy with analyses conducted at the molecular and cellular level. We identified that GH upregulates key mechanisms of therapy resistance and metastases in melanoma tumors in an IGF-1 dependent and independent manner by upregulating multidrug efflux pumps and EMT transcription factors. Our study reveals that GH action renders an intrinsic drug resistance phenotype to the melanoma tumors—a clinically crucial property of GH verifiable in other human cancers with GHR expression.Growth hormone (GH) and the GH receptor (GHR) are expressed in a wide range of malignant tumors including melanoma. However, the effect of GH/insulin-like growth factor (IGF) on melanoma in vivo has not yet been elucidated. Here we assessed the physical and molecular effects of GH on mouse melanoma B16-F10 and human melanoma SK-MEL-30 cells in vitro. We then corroborated these observations with syngeneic B16-F10 tumors in two mouse lines with different levels of GH/IGF: bovine GH transgenic mice (bGH; high GH, high IGF-1) and GHR gene-disrupted or knockout mice (GHRKO; high GH, low IGF-1). In vitro, GH treatment enhanced mouse and human melanoma cell growth, drug retention and cell invasion. While the in vivo tumor size was unaffected in both bGH and GHRKO mouse lines, multiple drug-efflux pumps were up regulated. This intrinsic capacity of therapy resistance appears to be GH dependent. Additionally, epithelial-to-mesenchymal transition (EMT) gene transcription markers were significantly upregulated in vivo supporting our current and recent in vitro observations. These syngeneic mouse melanoma models of differential GH/IGF action can be valuable tools in screening for therapeutic options where lowering GH/IGF-1 action is important.

RBC-derived vesicles as a systemic delivery system of doxorubicin for lysosomal-mitochondrial axis-improved cancer therapy

IntroductionChemotherapeutic drugs are the main intervention for cancer management, but many drawbacks impede their clinical applications. Nanoparticles as drug delivery systems (DDSs) offer much promise to solve these limitations. ObjectivesA novel nanocarrier composed of red blood cell (RBC)-derived vesicles (RDVs) surface-linked with doxorubicin (Dox) using glutaraldehyde (glu) to form Dox-gluRDVs was investigated for improved cancer therapy. MethodsWe investigated the in vivo antineoplastic performance of Dox-gluRDVs through intravenous (i.v.) administration in the mouse model bearing subcutaneous (s.c.) B16F10 tumor and examined the in vitro antitumor mechanism and efficacy in a panel of cancer cell lines. ResultsDox-gluRDVs can exert superior anticancer activity than free Dox in vitro and in vivo. Distinct from free Dox that is mainly located in the nucleus, but instead Dox-gluRDVs release and efficiently deliver the majority of their conjugated Dox into lysosomes. In vitro mechanism study reveals the critical role of lysosomal Dox accumulation-mediated mitochondrial ROS overproduction followed by the mitochondrial membrane potential loss and the activation of apoptotic signaling for superior anticancer activity of Dox-gluRDVs. ConclusionThis work demonstrates the great potential of RDVs to serve a biological DDS of Dox for systemic administration to improve conventional cancer chemotherapeutics.

A novel target anti-interleukin-13 receptor subunit alpha-2 monoclonal antibody inhibits tumor growth and metastasis in lung cancer

IL13Rα2 shows high expression in different types of tumors and can be a target for cancer therapy in humans due to its poor prognosis. The aim of our study is to characterize and investigate the effect of interleukin-13 receptor subunit alpha-2monoclonal antibody mAb15D8 on lung cancer cells in vitro and in vivo by blocking its specific epitope in IL13Rα2 antigen. The mAb15D8 blocking epitope was analyzed through the mutagenesis of IL13Rα2 and confirmed with western blot. We found that the IL13Rα2 epitope recognized by mAb15D8 antibody is a new binding site localized in the fibronectin-III domain-1 of IL13Rα2 antigen. Moreover, the mAb15D8 obviously reduced cell proliferation, migration of H460, A549, SKOV3, and B16F10 cells. Treatment with mAb15D8 significantly reduced the H460 xenograft tumor formation and growth in nude mice and inhibited B16F10 tumor metastasis and increased survival in C57BL/6 mice. Pharmacokinetic and toxicological analysis demonstrated the safety of mAb15D8 as a potential therapeutic agent. We developed a novel mouse monoclonal antibody against IL13Rα2 which binds to specific epitope on IL13Rα2 antigen. In vivo treatment with the antibody significantly reduced tumor growth and lung metastasis and prolonged survival. These results suggest mAb15D8 antibody as a potential therapeutic agent for cancer therapy.

Injectable Liquid Crystal Formation System for Reshaping Tumor Immunosuppressive Microenvironment to Boost Antitumor Immunity: Postoperative Chemoimmunotherapy.

Exploring optimal strategies to improve patient outcome postoperatively is still under challenge. Cancer immunotherapy has great potential to prevent the postoperative tumor recurrence and metastasis, which could be further strengthened by re-education of tumor microenvironment (TME). Herein, a local and sustained drug delivery system of liquid crystal formation system (LCFS) co-loaded with doxorubicin (DOX) and resiquimod (R848) (D/R@LCFS) is reported to confer effective chemoimmunotherapy with reduced systematic toxicity. After local administration, D/R@LCFS turns tumor into in situ vaccine via DOX-triggered immunogenic cell death effect accompanied with immunostimulatory effect of R848. Meanwhile, combination treatment of D/R@LCFS facilitates the recruitment of effector CD8+ T cells and the polarization of myeloid-derived suppressor cells and immunosuppressive type 2-polarized macrophages to tumoricidal antigen-presenting cells, favoring antigen-specific T cell immune response and inducing more immunogenic phenotypes in tumors. The generated in situ vaccine as well as reshaped TME by D/R@LCFS elicited systematic immune response and long term immune-memory effect in combination with immune checkpoint blockade to significantly prevent postoperative B16F10 or 4T1 tumor recurrence and metastasis. Therefore, this combination strategy of spatiotemporal TME modulation is expected to provide a clinical available option for effective postoperative chemoimmunotherapy.

Abstract PO-105: The fibrinolytic factor tPA drives LRP1-mediated melanoma growth and metastasis

Abstract The multifunctional endocytic receptor low-density lipoprotein receptor-related protein (LRP)1 has recently been identified as a hub within a biomarker network for multicancer clinical outcome prediction. The mechanismhowLRP1 modulates cancer progression is poorly understood. In this studywe found that LRP1 and one of its ligands, tissue plasminogen activator (tPA), are expressed inmelanoma cells and control melanoma growth and lung metastasis in vivo. Mechanistic studies were performed on 2 melanoma cancer cell lines, B16F10 and the B16F1 cells, both of which formprimary melanoma tumors, but only B16F10 cells metastasize to the lungs. Tumor-, but not niche cell–derived tPA, enhanced melanoma cell proliferation in tPA2/2 mice. Gain-of-function experiments revealed that melanoma LRP1 is critical for tumor growth, recruitment of mesenchymal stem cells into the tumor bed, and metastasis. Melanoma LRP1 was found to enhance ERK activation, resulting in increased matrix metal-loproteinase (MMP)-9 RNA, protein, and secreted activity, a well-known modulator of melanoma metastasis. Restoration of LRP1 and tPA in the less aggressive, poorly metastatic B16F1 tumor cells enhanced tumor cell proliferation and led to massive lung metastasis in murine tumor models. Antimelanoma drug treatment induced tPA and LRP1 expression. tPA or LRP1 knockdown enhanced chemosensitivity in melanoma cells. Our results identify the tPA-LRP1 pathway as a key switch that drives melanoma progression, in part by modulating the cellular composition and proteolytic makeup of the tumor niche. Targeting this pathway may be a novel treatment strategy in combination treatments for melanoma. Citation Format: Beate Heissig, Yousef Salama, Taro Osada, Yuko Tsuda, Satoshi Takahashi, Koichi Hattori. The fibrinolytic factor tPA drives LRP1-mediated melanoma growth and metastasis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-105.

Talin-dependent integrin activation is required for endothelial proliferation and postnatal angiogenesis.

Integrin activation contributes to key blood cell functions including adhesion, proliferation and migration. An essential step in the cell signaling pathway that activates integrin requires the binding of talin to the β-integrin cytoplasmic tail. Whereas this pathway is understood in platelets in detail, considerably less is known regarding how integrin-mediated adhesion in endothelium contributes to postnatal angiogenesis. We utilized an inducible EC-specific talin1 knock-out mouse (Tln1 EC-KO) and talin1 L325R knock-in mutant (Tln1 L325R) mouse, in which talin selectively lacks the capacity to activate integrins, to assess the role of integrin activation during angiogenesis. Deletion of talin1 during postnatal days 1-3 (P1-P3) caused lethality by P8 with extensive defects in retinal angiogenesis and widespread hemorrhaging. Tln1 EC-KO mice displayed reduced retinal vascular area, impaired EC sprouting and proliferation relative to Tln1 CTRLs. In contrast, induction of talin1 L325R in neonatal mice resulted in modest defects in retinal angiogenesis and mice survived to adulthood. Interestingly, deletion of talin1 or expression of talin1 L325R in ECs increased MAPK/ERK signaling. Strikingly, B16-F0 tumors grown in Tln1 L325R adult mice were 55% smaller and significantly less vascularized than tumors grown in littermate controls. EC talin1 is indispensable for postnatal development angiogenesis. The role of EC integrin activation appears context-dependent as its inhibition is compatible with postnatal development with mild defects in retinal angiogenesis but results in marked defects in tumor growth and angiogenesis. Inhibiting EC pan-integrin activation may be an effective approach to selectively target tumor blood vessel growth.

Upconversion optogenetic micro-nanosystem optically controls the secretion of light-responsive bacteria for systemic immunity regulation

Chemical molecules specifically secreted into the blood and targeted tissues by intestinal microbiota can effectively affect the associated functions of the intestine especially immunity, representing a new strategy for immune-related diseases. However, proper ways of regulating the secretion metabolism of specific strains still remain to be established. In this article, an upconversion optogenetic micro-nanosystem was constructed to effectively regulate the specific secretion of engineered bacteria. The system included two major modules: (i) Modification of secretory light-responsive engineered bacteria. (ii) Optical sensing mediated by upconversion optogenetic micro-nanosystem. This system could regulate the efficient secretion of immune factors by engineered bacteria through optical manipulation. Inflammatory bowel disease and subcutaneously transplanted tumors were selected to verify the effectiveness of the system. Our results showed that the endogenous factor TGF-β1 could be controllably secreted to suppress the intestinal inflammatory response. Additionally, regulatory secretion of IFN-γ was promoted to slow the progression of B16F10 tumor.

Phthalocyanine-based photosensitizers combined with anti-PD-L1 for highly efficient photodynamic immunotherapy

Immune checkpoint blockade (ICB) therapy combined with Photodynamic therapy (PDT) has shown great potential in the treatment of advanced metastatic tumors. However, it is still an important challenge to find a simple and effective photosensitizer for matching photodynamic immunotherapy. In this paper, we report a treatment method that combines PDT caused by three zinc(II) phthalocyanines (ZnPcs) with a different charge, and a checkpoint inhibition agent, namely anti-PD-L1 antibody, to induce systemic antitumor immunity. Among the ZnPcs, the neutral ZnPc 1 showed the highest photodynamic activity, while the cationic compound 2 exhibited the highest synergistic antitumor effect with ICB. For ZnPc 2, the tumor inhibition rate of PDT-ICB combined therapy for distant tumors was found to be as high as 89%, but PDT alone or ICB alone could hardly cause an inhibitory effect in a B16–F10 tumor model. We also detected an increase in T cell infiltration in the tumor microenvironment after the immune system was activated by a combination of anti-PD-L1 antibody and PDT. It was interesting to find that CD8+ T cells increased most significantly for the combination of anti-PD-L1 antibody and PDT by the positively charged compound 2. Moreover, the combination of ZnPc 2-PDT and ICB could efficiently inhibit the recurrence of tumors. The results suggest that the mono-cationic ZnPc 2 with a well-defined structure and ease of preparation, is a highly promising photosensitizer specifically for PDT combined with immunotherapy.

Polyamine Blocking Therapy Decreases Survival of Tumor-Infiltrating Immunosuppressive Myeloid Cells and Enhances the Antitumor Efficacy of PD-1 Blockade.

Despite unprecedented advances in the treatment of cancer through the use of immune checkpoint blockade (ICB), responses are not universal and alternative strategies are needed to enhance responses to ICB. We have shown previously that a novel polyamine blocking therapy (PBT), consisting of cotreatment with α-difluoromethylornithine (DFMO) to block polyamine biosynthesis and a Trimer polyamine transport inhibitor, decreases myeloid-derived suppressor cells (MDSC) and M2-like tumor-associated macrophages (TAM). Both MDSCs and TAMs promote tumor progression, inhibit antitumor immunity, and limit the efficacy of ICB. In this study, we investigated the use of PBT to heighten therapeutic responses to PD-1 blockade in mice bearing 4T1 mammary carcinoma and B16F10 melanoma tumors. Whereas PBT inhibited primary tumor growth in both tumor models, 4T1 lung metastases were also dramatically decreased in mice treated with PBT. Reductions in MDSC and TAM subpopulations in 4T1 tumors from PBT-treated mice were accompanied by reduced cytoprotective autophagy only in tumor-infiltrating MDSC and macrophage subpopulations but not in the lung or spleen. PBT treatment blunted M2-like alternative activation of bone marrow-derived macrophages and reduced STAT3 activation in MDSC cultures while increasing the differentiation of CD80+, CD11c+ macrophages. PBT significantly enhanced the antitumor efficacy of PD-1 blockade in both 4T1 and B16F10 tumors resistant to anti-PD-1 monotherapy, increasing tumor-specific cytotoxic T cells and survival of tumor-bearing animals beyond that with PBT or PD-1 blockade alone. Our results suggest that cotreatment with DFMO and the Trimer polyamine transport inhibitor may improve the therapeutic efficacy of immunotherapies in patients with cancer with resistant tumors.

SLAMF7 Signaling Reprograms T Cells Towards Exhaustion in the Tumor Microenvironment

T cell exhaustion represents one of the most pervasive strategies tumors employ to circumvent the immune system. While tonic T cell receptor (TCR) signaling is recognized as the primary driving force behind this phenomenon, it is still unknown what other forces drive T cell exhaustion in the tumor microenvironment. Here, using a combination of human in vitro and murine in vivo systems, we show that, independent of TCR signaling, the self-ligand SLAMF7 immune receptor drives the expression of both T cell inhibitory receptors and exhaustion-promoting transcription factors. SLAMF7 expression can be used to stratify clear cell renal cell carcinoma patients by survival, and T cell exhaustion in these patients is mediated by SLAMF7+ tumor-associated macrophages. Finally, mice lacking SLAMF7 show restricted growth of B16-F10 tumors and CD8+ T cells from these mice exhibited an impaired ability to progress through the exhaustion developmental trajectory to terminal exhaustion.

An implantable blood clot-based immune niche for enhanced cancer vaccination.

Cancer immunotherapy using cancer vaccines has shown great potential in the prevention and treatment of cancer. Here, we report an implantable autologous blood clot scaffold for enhanced cancer vaccination. It comprises a gel-like fibrin network formed by coagulation of blood to trap a large number of red blood cells. Upon implantation, the cross-linked RBCs in the blood clot can attract and recruit a great number of immune cells, leading to the formation of an "immune niche." Encapsulated with tumor-associated antigen and adjuvant, the blood clot vaccine (BCV) can induce a robust anticancer immune response. The BCV combined with immune checkpoint blockade effectively inhibits tumor growth in B16F10 and 4T1 tumor models. The proposed implantable blood clot cancer vaccine can be readily made by mixing the blood from patients with cancer with immunomodulating agents ex vivo, followed by reimplantation into the same patient for personalized cancer immunotherapy in future clinical translation.

IL-32γ potentiates tumor immunity in melanoma.

Myeloid cells orchestrate the antitumor immune response and influence the efficacy of immune checkpoint blockade (ICB) therapies. We and others have previously shown that IL-32 mediates DC differentiation and macrophage activation. Here, we demonstrate that IL-32 expression in human melanoma positively correlates with overall survival, response to ICB, and an immune-inflamed tumor microenvironment (TME) enriched in mature DC, M1 macrophages, and CD8+ T cells. Treatment of B16F10 murine melanomas with IL-32 increased the frequencies of activated, tumor-specific CD8+ T cells, leading to the induction of systemic tumor immunity. Our mechanistic in vivo studies revealed a potentially novel role of IL-32 in activating intratumoral DC and macrophages to act in concert to prime CD8+ T cells and recruit them into the TME through CCL5. Thereby, IL-32 treatment reduced tumor growth and rendered ICB-resistant B16F10 tumors responsive to anti–PD-1 therapy without toxicity. Furthermore, increased baseline IL-32 gene expression was associated with response to nivolumab and pembrolizumab in 2 independent cohorts of patients with melanoma, implying that IL-32 is a predictive biomarker for anti–PD-1 therapy. Collectively, this study suggests IL-32 as a potent adjuvant in immunotherapy to enhance the efficacy of ICB in patients with non–T cell–inflamed TME.

Inactivation of endothelial cell phosphoinositide 3-kinase β inhibits tumor angiogenesis and tumor growth.

Angiogenesis inhibitors, such as the receptor tyrosine kinase (RTK) inhibitor sunitinib, target vascular endothelial growth factor (VEGF) signaling in cancers. However, only a fraction of patients respond, and most ultimately develop resistance to current angiogenesis inhibitor therapies. Activity of alternative pro-angiogenic growth factors, acting via RTK or G-protein coupled receptors (GPCR), may mediate VEGF inhibitor resistance. The phosphoinositide 3-kinase (PI3K)β isoform is uniquely coupled to both RTK and GPCRs. We investigated the role of endothelial cell (EC) PI3Kβ in tumor angiogenesis. Pro-angiogenic GPCR ligands were expressed by patient-derived renal cell carcinomas (PD-RCC), and selective inactivation of PI3Kβ reduced PD-RCC-stimulated EC spheroid sprouting. EC-specific PI3Kβ knockout (ΕC-βKO) in mice potentiated the sunitinib-induced reduction in subcutaneous growth of LLC1 and B16F10, and lung metastasis of B16F10 tumors. Compared to single-agent sunitinib treatment, tumors in sunitinib-treated ΕC-βKO mice showed a marked decrease in microvessel density, and reduced new vessel formation. The fraction of perfused mature tumor microvessels was increased in ΕC-βKO mice suggesting immature microvessels were most sensitive to combined sunitinib and PI3Kβ inactivation. Taken together, EC PI3Kβ inactivation with sunitinib inhibition reduces microvessel turnover and decreases heterogeneity of the tumor microenvironment, hence PI3Kβ inhibition may be a useful adjuvant antiangiogenesis therapy with sunitinib.

Host genetics and tumor environment determine the functional impact of neutrophils in mouse tumor models.

BackgroundNeutrophils have been reported to have protumor, antitumor or neutral effects in cancer progression. The underlying causes for this functional variability are not clear.MethodsWe studied the role of neutrophils in six different mouse tumor models by intratumoral injection of antimicrobial peptides or vaccination. Changes in systemic and intratumoral immune cells were analyzed by flow-cytometry and mass-cytometry. The role of neutrophils was studied by antibody-mediated neutrophil depletion. Neutrophils from different mouse strains were compared by RNA sequencing.ResultsThe antimicrobial peptide Omiganan reduced the growth of TC-1 tumors in BL/6 mice and CT26 tumors in BALB/c mice. No significant effects were observed in B16F10, MC38 and 4T1 tumors. Growth delay was associated with increased abundance of neutrophils in TC-1 but not CT26 tumors. Systemic neutrophil depletion abrogated Omiganan efficacy in TC-1 but further reduced growth of CT26, indicating that neutrophils were required for the antitumor effect in TC-1 but suppressed tumor control in CT26. Neutrophils were also required for a therapeutic vaccine-induced T-cell mediated control of RMA tumors in BL/6 mice. Clearly, the circulating and intratumoral neutrophils differed in the expression of Ly6G and CD62L, between TC-1 and CT26 and between blood neutrophils of tumor-naïve BL/6 and BALB/c mice. RNA-sequencing revealed that neutrophils from BL/6 mice but not BALB/c mice displayed a robust profile of immune activation, matching their opposing roles in TC-1 and RMA versus CT26.ConclusionsNeutrophil functionality differs strongly between mouse strains and tumor types, with consequences for tumor progression and therapy.

An Analysis of Isoclonal Antibody Formats Suggests a Role for Measuring PD-L1 with Low Molecular Weight PET Radiotracers.

The swell of new and diverse radiotracers to predict or monitor tumor response to cancer immunotherapies invites the opportunity for comparative studies to identify optimal platforms. To probe the significance of antibody format on image quality for PD-L1 imaging, we developed and studied the biodistribution of a library of antibodies based on the anti-PD-L1 IgG1 clone C4. A C4 minibody and scFv were cloned, expressed, and characterized. The antibodies were functionalized with desferrioxamine and radiolabeled with Zr-89 to enable a rigorous comparison with prior data collected using 89Zr-labeled C4 IgG1. The biodistribution of the radiotracers was evaluated in C57Bl6/J or nu/nu mice bearing B16F10 or H1975 tumors, respectively, which are models that represent high and low tumor autonomous PD-L1 expression. The tumor uptake of the 89Zr-C4 minibody was higher than 89Zr-C4 scFv and equivalent to previous data collected using 89Zr-C4 IgG1. However, the peak tumors to normal tissue ratios were generally higher for 89Zr-C4 scFv compared with 89Zr-C4 minibody and 89Zr-IgG1. Moreover, an exploratory study showed that the rapid clearance of 89Zr-C4 scFv enabled detection of endogenous PD-L1 on a genetically engineered and orthotopic model of hepatocellular carcinoma. In summary, these data support the use of low molecular weight constructs for PD-L1 imaging, especially for tumor types that manifest in abdominal organs that are obstructed by the clearance of high molecular weight radioligands.