B16F10 Tumor Research Articles

Abstract P060: Identification of host-intrinsic resistance mechanisms to immune checkpoint inhibitors (ICI) in Diversity Outbred mice

Abstract The advent of immune checkpoint inhibitors (ICI) for cancer therapy has improved outcomes for a variety of malignancies, however many a significant proportion of patients fail to benefit. While tumor-intrinsic mechanisms are likely involved in therapy resistance, it is unclear to what extent genetic heterogeneity of the host plays in response. To investigate this in a pre-clinical setting, we utilize the Diversity Outbred (DO) and Collaborative Cross (CC) mouse models. DO mice are genetically distinct animals generated by cross-breeding 8 inbred founder strains, and CC mice are recombinant inbred mice generated from the same 8 founders as DO. We find a wide variation in response to anti-PD-1/anti-CTLA-4 ICI in B16F0-bearing (C57BL/6xDO)F1 mice (n=142) with ~30% showing partial response with delayed tumor onset and ~13% showing complete tumor elimination with protection against subsequent contralateral rechallenge. Genetic linkage analysis using the R/qtl2 package identifies genomic loci associated with therapeutic outcomes. Importantly, the MHC locus was not significantly associated with response. Of the strongly associated loci, the region within chromosome 13 was further validated in CC mice bearing the positive (NZO) versus negative (C57BL/6) driver genotypes at this locus. ICI response was detected in 2/3 (C57BL/6xCC)F1 predicted responder models, with no response in predicted non-responders. This locus solely contains the murine prolactin family. Prolactin is a known immunomodulating cytokine with association to various autoimmune disorders. To directly test whether prolactin influences ICI response rates, we implanted inbred C57BL/6 mice, which notoriously fail to respond to ICI alone against B16F0 tumors, with subcutaneous slow-release prolactin pellets to induce mild hyperprolactinemia. Combined ICI with prolactin shows an interaction effect against B16F0, with 5/8 mice exhibiting slowed tumor growth relative to controls. To test whether this host-derived phenomenon applies to additional tumor models, we tested ICI response to Lewis Lung Carcinoma (LLC) in a (C57BL/6xCC)F1 model where response to B16F0 was observed. Relative to inbred C57BL/6 mice, where no response to ICI is observed, this (C57BL/6xCC)F1 model shows a similar response to ICI against LLC as was detected for B16F0. Collectively, this study shows that host genetics play a role in ICI response and that prolactin may be a positive regulator of ICI efficacy. Citation Format: Justin Hackett, James Glassbrook, Maria Muniz, Heather M. Gibson. Identification of host-intrinsic resistance mechanisms to immune checkpoint inhibitors (ICI) in Diversity Outbred mice [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P060.

Hinokitiol impedes tumor drug resistance by suppressing protein kinase B/mammalian targets of rapamycin axis.

Chemotherapy is a treatment method commonly used for cancer and that patients showing low to no response to the treatment often developed drug resistance via multiple mechanisms. Natural products have been shown to reduce tumor drug resistance. Hinokitiol, a natural tropolone derivative, has potential as an antitumor agent. To improve the efficacy and safety of hinokitiol, a further understanding of hinokitiol interactions with the tumor microenvironment is necessary. The presence of plasma membrane multidrug resistance protein P-glycoprotein (P-gp) is favorable for tumor cells to elicit chemotherapeutic resistance. Here, we showed that hinokitiol dose-dependently decreased P-gp expression and suppressed the P-gp-driven efflux activity based on Rhodamine 123 assay. The protein expression levels of phosph-protein kinase B (P-AKT), phosph-mammalian targets of rapamycin (P-mTOR), and phosph-p70 ribosomal s6 kinase (P-p70s6K) in tumor cells were likewise reduced after hinokitiol treatment. The transfection of cells with active P-AKT rescued hinokitiol-induced downregulation of P-gp, suggesting the involvement of Akt/mTOR/p70s6K signaling in P-gp expression. Our results showed that hinokitiol can chemosensitize cancer cells. These findings indicate that hinokitiol could enhance 5-Fluorouracil therapeutic effects in murine B16F10 and CT26 tumor cells via downregulation of the AKT/mTOR pathway.

Paclitaxel treatment enhances lymphatic metastasis of B16F10 melanoma cells via CCL21/CCR7 axis.

Chemotherapeutic drugs have been successfully used to treat several cancers, including melanoma. However, metastasis occasionally occurs after chemotherapy. Here, we reported that paclitaxel (PTX) treatment for B16F10 tumour in mice led to an enhanced lymphatic metastasis of the melanoma cells, although a significant inhibition of tumour growth at the injection site was observed. Further study demonstrated that PTX upregulated the expression of C-C chemokine receptor type 7 (CCR7) in B16F10 cells, enhancing their migration through the activation of JNK and p38 signalling pathways. Loss of CCR7 or blockade of C-C motif chemokine ligand 21 (CCL21)/CCR7 axis abolished the pro-migration effect of PTX on B16F10 melanoma cells. Importantly, combination of PTX and CCR7 mAb could simultaneously delay the tumour growth and reduce the lymphatic metastasis in B16F10 melanoma. The blockade of CCL21/CCR7 axis may collectively serve as a strategy for lymphatic metastasis in some melanoma after chemotherapy.

Abstract P006: Intratumoral electroporation of IL-12 and SARS-Cov-2 spike plasmids drives a coordinated vaccine response and elicits robust anti-tumor immunity

Abstract Despite extensive clinical evidence on the efficacy and safety of SARS-CoV-2 vaccines, there remains a paucity of data on their effectiveness in cancer patients who are actively receiving antineoplastic therapeutics. A recent study demonstrated only ~30% of cancer patients had positive serologic test following 2 doses of FDA-authorized SARS-CoV-2 vaccines, in contrast to ~80% positivity rate in healthy individuals, regardless of the age. Therefore, further investigation into novel approaches to boost immune response to SARS-CoV-2 vaccines in cancer patients is required. Our previous preclinical and clinical studies have established intratumoral IL-12 plasmid (TAVO) electroporation (EP) induces localized expression of IL-12p70, converting immune-excluded tumors into inflamed immunogenic lesions, thereby generating objective responses in both treated and untreated, distant tumors. Based on the enhancement of immunotherapy efficacy by IL-12, we leveraged the flexibility of our DNA plasmid-EP platform to express SARS-CoV-2 spike protein in addition to IL-12 (CORVax12) as an intratumoral vaccine candidate which we hypothesized could not only drive anti-SARS-CoV-2 immune responses but also generate a productive anti-tumor response. Naïve mice were vaccinated via intradermal injection of SARS-CoV-2 spike plasmid followed immediately by EP with or without plasmid-encoded mIL-12 on days 1 and 21. Longitudinal serum samples were collected to interrogate virus-specific cellular responses as well anti-spike IgG antibody. A surrogate viral neutralization test (sVNT) assessed serum blockade of soluble human ACE2 binding to immobilized SARS-CoV-2 spike. Our data demonstrated that intradermally electroporated CORVax12 elicits significantly higher anti-SARS-CoV-2 spike IgG antibodies and neutralization when compared with EP of SARS-CoV-2 spike alone. Next, we asked if improved SARS-CoV-2 immune response may be observed when CORVax12 is incorporated into intratumoral EP in single-tumor bearing mice. CORVax12 robustly inhibited tumor growth, induced high percentages of germinal-center B cells and class switched B cells in tumor draining lymph nodes, and generated high of anti-spike IgG and neutralization antibodies. To further investigate systemic effects of this combination, we continued with contralateral tumor mice models. In both CT26 and B16-F10 tumor models, CORVax12 intratumoral EP induced strong systemic anti-tumor responses similar to IL-12 EP alone while also producing high serum levels of anti-SARS-CoV-2 spike IgG and neutralization antibodies. Critically, this anti-viral immunity did not limit this IL-12-based intratumoral anti-tumor therapy. In summary, our preclinical data indicates that intratumoral EP of CORVax12 can induce IgG responses to SARS-CoV-2 spike as well as apparent viral neutralizing activity all while maintaining local and systemic anti-tumor effects expected from TAVO Treatment. This combined intratumoral therapy represents a novel strategy to address both tumor burden and anti-SARS-CoV-2 immunity in patients with cancer. Citation Format: Mia Han, Jack Y. Lee, Vincent Wu, Kurt Sakurada, Bianca Nguyen, David A. Canton, Christopher G. Twitty. Intratumoral electroporation of IL-12 and SARS-Cov-2 spike plasmids drives a coordinated vaccine response and elicits robust anti-tumor immunity [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P006.

Mild hyperthermia accelerates doxorubicin clearance from tumour-extravasated temperature-sensitive liposomes.

Mild hyperthermia (HT) (40-43 °C) has been combined with temperature-sensitive liposomes (TSL), offering on-demand drug release for increased drug bioavailability and reduced systemic toxicity. Different HT regimens have been applied to trigger liposome drug release in the blood vessels (intravascular) of heated tumours or following tumour extravasation (interstitial). The present study systematically assessed the in vivo doxorubicin (Dox) release and therapeutic efficacy of Dox-loaded TSL with different release profiles. Low temperature-sensitive liposomes (LTSL-Dox), traditional-temperature-sensitive liposomes (TTSL-Dox), and non-temperature-sensitive liposomes (NTSL-Dox) were combined with a single or two HT in different tumour models (murine melanoma B16F10 tumour and human breast MDA-MB-435). The efficacy of each treatment was assessed by monitor tumour growth and mice survival. The level of Dox in tumour tissues was quantified using 14C-Dox and liquid scintillation while Dox release was assessed using live imaging and confocal laser scanning microscopy. Applying a second HT to release Dox from extravasated TTSL-Dox was not therapeutically superior to single HT application due to Dox clearance from the extravasated TTSL-Dox. Our findings revealed that enhanced blood perfusion in heated tumours during the second water bath HT could be seen as a hurdle for TTSL-Dox's anticancer efficacy, where the systemic toxicity of the redistributed Dox from the tumour tissues could be potentiated.

A Novel Small Cyclic Peptide-Based 68Ga-Radiotracer for Positron Emission Tomography Imaging of PD-L1 Expression in Tumors.

In the tumor microenvironment, programmed death protein 1 and programmed death protein ligand 1 (PD-L1) signaling pathways help tumors escape the immune system. We designed a gallium-68 (68Ga)-labeled small-molecule peptide-targeting PD-L1 and used positron emission tomography/computed tomography (PET/CT) to detect and dynamically monitor the expression level of PD-L1 in tumors. S-Cyclo(ETSK)-SF-NH2 (SETSKSF) is a cyclic peptide inhibitor comprising seven amino-acid residues. We connected it with the chelating agent DOTA, labeled DOTA-SETSKSF, with the short half-life nuclide Ga-68, and measured the stability of 68Ga-2,2',2″-(10-(2-((S)-1-((3S,6S,9S,18S)-18-((S)-1-((S)-1-amino-1-oxo-3-henylpropan-2-ylamino)-3-hydroxy-1-oxopropan-2-ylcarbamoyl)-6-((R)-1-hydroxyethyl)-3-(hydroxymethyl)-2,5,8,12-tetraoxo-1,4,7,13-tetraazacyclooctadecan-9-ylamino)-3-ydroxy-1-oxopropan-2-ylamino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (68Ga-DOTA-SETSKSF) in normal saline (NS), phosphate-buffered saline (PBS), and fetal bovine serum (FBS) in vitro. We conducted the 68Ga-DOTA-SETSKSF affinity test, cell-specific uptake experiments, time-combined experiments, western blotting, and laser confocal experiments to confirm the expression and localization of PD-L1 at the cell level and determine the uptake. Biodistribution and imaging experiments were performed using the H1975, B16F10, and A549 tumor models. 68Ga-DOTA-SETSKSF was successfully synthesized, and the radiochemical purity was >99% after purification. The in vitro stability of 68Ga-DOTA-SETSKSF was >95% in NS, PBS, and FBS at 37 °C after 4 h of incubation. Cell-binding experiments confirmed that 68Ga-DOTA-SETSKSF exhibited high uptake in H1975 tumors with high PD-L1 expression and low uptake in A549 tumors with low PD-L1 expression. The clear half-life (T1/2) of 68Ga-DOTA-SETSKSF from the blood was 14.48 ± 3.26 min. The percentages of the injected dose per gram of tissue (%ID/g) for H1975 and A549 tumors were 5.29 ± 0.21 and 0.89 ± 0.10 at 1 h after injection, respectively. The H1975 tumor-to-muscle and tumor-to-blood ratios were 41.79 ± 5.81 and 4.75 ± 0.19 at 4 h, respectively. Apart from the H1975 tumor, the kidney and the bladder showed high accumulation because 68Ga-DOTA-SETSKSF was excreted through the urinary system. PET/CT images showed high accumulation of 68Ga-DOTA-SETSKSF in H1975 tumors and low uptake in A549 tumors, which was consistent with the results of biodistribution experiments. 68Ga-DOTA-SETSKSF is convenient to prepare, has high stability, can be used to monitor the expression of PD-L1, and has an extremely high clinical application value.

Cancer-Erythrocyte Hybrid Membrane-Camouflaged Magnetic Nanoparticles with Enhanced Photothermal-Immunotherapy for Ovarian Cancer

Cell-membrane-coated nanoparticles are widely studied due to their inherent cellular properties, such as immune escape and homologous homing. A cell membrane coating can also maintain the relative stability of nanoparticles during circulation in a complex blood environment through cell membrane encapsulation technology. In this study, we fused a murine-derived ID8 ovarian cancer cell membrane with a red blood cell (RBC) membrane to create a hybrid biomimetic coating (IRM), and hybrid IRM camouflaged indocyanine green (ICG)-loaded magnetic nanoparticles (Fe3O4-ICG@IRM) were fabricated for combination therapy of ovarian cancer. Fe3O4-ICG@IRM retained both ID8 and RBC cell membrane proteins and exhibited highly specific self-recognition of ID8 cells in vitro and in vivo as well as a prolonged circulation lifetime in blood. Interestingly, in the bilateral flank tumor model, the IRM-coated nanoparticles also activated specific immunity, which killed homologous ID8 tumor cells but had no effect on B16-F10 tumor cells. Furthermore, Fe3O4-ICG@IRM showed synergistic photothermal therapy, resulting in the release of whole-cell tumor antigens by photothermal-induced tumor necrosis, which further enhanced antitumor immunotherapy for primary tumor and metastatic tumor by activating CD8+ cytotoxic T cells and reducing regulatory Foxp3+ T cells. Together, the biomimetic Fe3O4-ICG@IRM nanoparticles showed synergistic photothermal-immunotherapy for ovarian cancer.

Biomimetic nanoparticles deliver mRNAs encoding costimulatory receptors and enhance T cell mediated cancer immunotherapy

Antibodies targeting costimulatory receptors of T cells have been developed for the activation of T cell immunity in cancer immunotherapy. However, costimulatory molecule expression is often lacking in tumor-infiltrating immune cells, which can impede antibody-mediated immunotherapy. Here, we hypothesize that delivery of costimulatory receptor mRNA to tumor-infiltrating T cells will enhance the antitumor effects of antibodies. We first design a library of biomimetic nanoparticles and find that phospholipid nanoparticles (PL1) effectively deliver costimulatory receptor mRNA (CD137 or OX40) to T cells. Then, we demonstrate that the combination of PL1-OX40 mRNA and anti-OX40 antibody exhibits significantly improved antitumor activity compared to anti-OX40 antibody alone in multiple tumor models. This treatment regimen results in a 60% complete response rate in the A20 tumor model, with these mice being resistant to rechallenge by A20 tumor cells. Additionally, the combination of PL1-OX40 mRNA and anti-OX40 antibody significantly boosts the antitumor immune response to anti-PD-1 + anti-CTLA-4 antibodies in the B16F10 tumor model. This study supports the concept of delivering mRNA encoding costimulatory receptors in combination with the corresponding agonistic antibody as a strategy to enhance cancer immunotherapy.

DDRE-01. CDK PATHWAY INHIBITION WITH ABEMACICLIB IMPROVES INTRACRANIAL AND EXTRACRANIAL RESPONSE TO CHECKPOINT BLOCKADE IN PRE-CLINICAL MODELS OF MELANOMA BRAIN METASTASIS

Abstract While immune checkpoint inhibitors (ICI) have revolutionized treatment of metastatic melanoma, few of the 75% of patients who develop brain metastases benefit from immunotherapy. Inhibition of CDK4/6 pathway – altered in ~90% of melanoma patients – can reportedly increase tumor inflammation and sensitize extracranial tumors to ICI. To determine whether intracranial melanoma can be similarly sensitized, we studied efficacy of combination CDK4/6 inhibitor Abemaciclib and ICI in immunocompetent mouse models of melanoma brain metastases bearing concurrent intracranial and extracranial tumors. 8-week-old female C57BL/6 mice received subcutaneous injections of 2x105 YUMM1.7 or B16-F10 melanoma cells 3 days prior to intracranial injections of 5x104 YUMM1.7 cells or 5x103 B16-F10 cells respectively. Mice were randomized into 6 treatment groups (n=5-7/group): Abemaciclib alone, anti-PD-1 monotherapy, anti-PD-1 and anti-CTLA4 combined (combination ICI), Abemaciclib and anti-PD-1, Abemaciclib combined with anti-PD-1 and anti-CTLA4 (triple therapy), and treatment with vehicle and isotype-matched antibodies as control. In mice bearing YUMM1.7 tumors, subcutaneous tumor growth was significantly reduced compared to control in mice treated with Abemaciclib alone (p< 0.05), combination ICI (p< 0.05) and triple therapy (p< 0.05). However, improvement in survival was only observed with triple therapy (p=0.039) compared to control group. In mice bearing B16-F10 tumors, we observed striking reduction in subcutaneous tumor growth in mice treated with Abemaciclib and anti-PD-1 compared to control-treated mice (p=0.0016) or to mice receiving anti-PD-1 monotherapy (p=0.000056). This further corresponded to a significant increase in survival of Abemaciclib and anti-PD-1 treated mice compared to control (p=0.02). Additionally, we observed improved survival in mice treated with combination ICI (p=0.006) or triple therapy (p=0.01). These results indicate CDK4/6 inhibition with Abemaciclib can improve both extracranial and intracranial responses to ICI and sensitize melanoma brain metastases to immunotherapy. Our pre-clinical findings warrant further investigation to determine whether this combination approach can improve patient outcomes.

Intratumoral DNA-based delivery of checkpoint-inhibiting antibodies and interleukin 12 triggers T cell infiltration and anti-tumor response.

To improve the anti-tumor efficacy of immune checkpoint inhibitors, numerous combination therapies are under clinical evaluation, including with IL-12 gene therapy. The current study evaluated the simultaneous delivery of the cytokine and checkpoint-inhibiting antibodies by intratumoral DNA electroporation in mice. In the MC38 tumor model, combined administration of plasmids encoding IL-12 and an anti-PD-1 antibody induced significant anti-tumor responses, yet similar to the monotherapies. When treatment was expanded with a DNA-based anti-CTLA-4 antibody, this triple combination significantly delayed tumor growth compared to IL-12 alone and the combination of anti-PD-1 and anti-CTLA-4 antibodies. Despite low drug plasma concentrations, the triple combination enabled significant abscopal effects in contralateral tumors, which was not the case for the other treatments. The DNA-based immunotherapies increased T cell infiltration in electroporated tumors, especially of CD8+ T cells, and upregulated the expression of CD8+ effector markers. No general immune activation was detected in spleens following either intratumoral treatment. In B16F10 tumors, evaluation of the triple combination was hampered by a high sensitivity to control plasmids. In conclusion, intratumoral gene electrotransfer allowed effective combined delivery of multiple immunotherapeutics. This approach induced responses in treated and contralateral tumors, while limiting systemic drug exposure and potentially detrimental systemic immunological effects.

Reprogramming NK cells and macrophages via combined antibody and cytokine therapy primes tumors for elimination by checkpoint blockade.

SUMMARYTreatments aiming to augment immune checkpoint blockade (ICB) in cancer often focus on T cell immunity, but innate immune cells may have important roles to play. Here, we demonstrate a single-dose combination treatment (termed AIP) using a pan-tumor-targeting antibody surrogate, half-life-extended interleukin-2 (IL-2), and anti-programmed cell death 1 (PD-1), which primes tumors to respond to subsequent ICB and promotes rejection of large established tumors in mice. Natural killer (NK) cells and macrophages activated by AIP treatment underwent transcriptional reprogramming; rapidly killed cancer cells; governed the recruitment of cross-presenting dendritic cells (DCs) and other leukocytes; and induced normalization of the tumor vasculature, facilitating further immune infiltration. Thus, innate cell-activating therapies can initiate critical steps leading to a self-sustaining cycle of T cell priming driven by ICB.

A novel form of immunotherapy using antigen peptides conjugated on PD-L1 antibody

Immune checkpoint inhibitors (ICIs), including programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 have shown promising cancer clinical outcomes. However, IC therapy has low patient response rates (10%–15%). Thus, ICIs and sufficient antigen combinations into the tumor microenvironment (TME) is important to produce strong tumor-specific adaptive immune responses. Mice were treated with cisplatin, and human cancer cells were exposed to inflammatory cytokines, to confirm increased PD-L1 and major histocompatibility complex (MHC) I expression by tumor cells or dendritic cells. TC-1, CT26, B16-F1, or B16-F10 tumor cells, and bone marrow-derived dendritic cells, were treated with interferon (IFN)-β, IFN-γ, or tumor necrosis factor-α to identify the molecular mechanisms underlying tumor PD-L1 and MHC I upregulation, and to examine MHC I, CD40, CD80, CD86, or PD-L1 levels, respectively. For synergistic combination therapy, αPD-L1 monoclonal antibody (mAb) covalently linked to the long E7 peptide was generated. Chemotherapy shifted the TME to express high PD-L1 and MHC I, resulting in targeted ICI cargo delivery and enhanced generation and activation of tumor antigen-specific T cells. Synergistic effects of vaccination and IC blockade in the TME were demonstrated using an anti-PD-L1 mAb covalently conjugated to the E7 long peptide.

Senolytic Cocktail Dasatinib Plus Quercetin Enhances the Antitumor Effect of Senescence-Inducing Radiotherapy in a Preclinical Model of Melanoma

Senolytic Cocktail Dasatinib Plus Quercetin Enhances the Antitumor Effect of Senescence-Inducing Radiotherapy in a Preclinical Model of Melanoma

MHC-Optimized Peptide Scaffold for Improved Antigen Presentation and Anti-Tumor Response.

Tumor Associated Antigens (TAAs) may suffer from an immunological tolerance due to expression on normal cells. In order to potentiate their immunogenicity, heteroclitic peptides (htcPep) were designed according to prediction algorithms. In particular, specific modifications were introduced in peptide residues facing to TCR. Moreover, a MHC-optimized scaffold was designed for improved antigen presentation to TCR by H-2Db allele. The efficacy of such htcPep was assessed in C57BL/6 mice injected with syngeneic melanoma B16F10 or lung TC1 tumor cell lines, in combination with metronomic chemotherapy and immune checkpoint inhibitors. The immunogenicity of htcPep was significantly stronger than the corresponding wt peptide and the modification involving both MHC and TCR binding residues scored the strongest. In particular, the H-2Db-specific scaffold significantly potentiated the peptides’ immunogenicity and control of tumor growth was comparable to wt peptide in a therapeutic setting. Overall, we demonstrated that modified TAAs show higher immunogenicity compared to wt peptide. In particular, the MHC-optimized scaffold can present different antigen sequences to TCR, retaining the conformational characteristics of the corresponding wt. Cross-reacting CD8+ T cells are elicited and efficiently kill tumor cells presenting the wild-type antigen. This novel approach can be of high clinical relevance in cancer vaccine development.

Synergy between Toxoplasma gondii type I ΔGRA17 immunotherapy and PD-L1 checkpoint inhibition triggers the regression of targeted and distal tumors

BackgroundIn this study, we hypothesize that the ability of the protozoan Toxoplasma gondii to modulate immune response within the tumor might improve the therapeutic effect of immune checkpoint blockade. We...

Blockade of TGF-βR improves the efficacy of doxorubicin by modulating the tumor cell motility and affecting the immune cells in a melanoma model.

TGF-β contributes to drug resistance and the invasiveness of tumor cells and weakens the anti-tumor immune responses. The present study aimed at examining the efficacy of the combination of SB431542, as a specific inhibitor of TGF-βR, and doxorubicin in controlling the melanoma tumor in mice. The impact of the combination of the doxorubicin and SB431542 on the cell growth, apoptosis, migration, and invasiveness of B16-F10 cells was examined. Besides, the B16-F10 tumor was induced in C57BL/6 mice, and the effects of the mentioned treatment on the tumor volume, survival, and the exhaustion state of T cells were evaluated. Although the combination of doxorubicin and SB431542 did not exhibit synergism in the inhibition of cell growth and apoptosis induction, it efficiently prohibited the migration and the epithelial to mesenchymal transition of B16-F10 cells, and the combination of doxorubicin and SB431542 caused an increase in mRNA levels of E-cadherin and, on the other hand, led to a decline in the expression of Vimentin. Tumor volume and the survival of tumor-bearing mice were efficiently controlled by the combination therapy. This treatment also eventuated in a decrease in the percentage of PD-L1+, TCD4+, and TCD8+ cells as indicators of exhausted T cells within the spleens of tumor-bearing mice. Blockade of TGF-βR also propelled the RAW 264.7 cells towards an anti-tumor M1 macrophage phenotype. The inhibition of TGF-βR demonstrated a potential to increase the efficacy of doxorubicin chemotherapy by the means of affecting cellular motility and restoring the anti-tumor immune responses.

Effects of exercise and anti-PD-1 on the tumour microenvironment

Immune checkpoint inhibition is highly effective in treating a subset of patients with certain cancers, such as malignant melanoma. However, a large proportion of patients will experience treatment resistance, and other tumour types, such as breast cancer, have thus far proven largely refractory to immune checkpoint inhibitors as single agents. Exercise has been associated with improved cancer patient survival, has known immune-modulatory effects, may improve anti-tumour immunity and may normalise tumour blood vessels. Therefore, we hypothesised that post-implant exercise would boost the effect of concurrent immunotherapy by enhancing anti-tumour immune responses and improving tumour blood flow. To investigate this, mice with EO771 breast tumours or B16-F10 melanomas received anti-PD-1, an isotype control antibody or no treatment. Mice were randomised to exercise (voluntary wheel running) or no exercise at tumour implant. Exercise reduced the number of CD8+T cells in EO771 (p = 0.0011) but not B16-F10 tumours (p = 0.312), and reduced the percentage of CD8+T cells within the total T cell population in both tumour types (B16-F10: p = 0.0389; EO771: p = 0.0015). In contrast, the combination of exercise and anti-PD-1 increased the percentage of CD8+T cells in EO771 (p = 0.0339) but not B16-F10 tumours. Taken together, our results show that exercise and anti-PD-1 induce changes in the tumour immune microenvironment which are dependant on tumour type.

A Sampling of Highlights from the Literature: Article Recommendations from Our Deputy and Senior Editors.

Modulating RNA splicing can augment antitumor T-cell responses (by Srosenbe via Wikimedia Commons)Altered RNA splicing in cancer cells can give rise to neoantigens, but it is unclear whether this is clinically relevant and therapeutically targetable. Using B16-F10 and MC38 tumor models, Lu et al. find several compounds that modulate RNA splicing can inhibit tumor growth and improve responses to anti-PD1 checkpoint blockade. These effects are dependent on T cells and MHC class I peptide presentation. Neoepitopes induced by pharmacologic modulation of RNA splicing in B16-F10 cells can trigger antitumor T-cell responses in mice. The data suggest potential new approaches to enhancing cancer immunotherapy.Lu SX, …, Bradley RK. Cell 2021 Jul 22;184:4032–47.e31.Arming CAR T cells with BATF enhances efficacy (from Spezadams via Wikimedia Commons)The transcriptional networks underlying T-cell exhaustion, including exhaustion in CAR T cells, are not fully known. Seo et al. show that basic leucine zipper ATF-like transcription factor (BATF) and interferon regulatory factor 4 (IRF4) act in a cooperative manner to offset T-cell exhaustion. Overexpression of BATF in CAR T cells increases cell persistence and cytokine production and decreases expression of inhibitory receptors and exhaustion-related markers. These effects are dependent on interactions between BATF and IRF4. The data highlight potential therapeutic targets to improve CAR T-cell therapy.Seo H, …, Hogan PG. Nat Immunol 2021 Aug 1;22:983–95.TILs with differing specificities have different functional states (from Brandon Dilbeck via Wikimedia Commons)Understanding of the relationship between the functional state of tumor-infiltrating lymphocytes (TIL) and the specificity of their TCRs is limited, but advances in single-cell technology are changing this. Using such technologies, Caushi et al. show that non–small cell lung cancer TILs with TCRs specific for mutation-associated neoantigens (MANA) have a unique transcriptional profile that is largely characteristic of tissue-resident memory cells and that MANA-specific T cells from tumors nonresponsive to anti-PD1 express significantly reduced levels of genes associated with T-cell dysfunction. Oliveira et al. show that melanoma-reactive TILs are predominantly in an exhausted state and only rarely acquire memory properties, and that disease persistence correlates with persistence of exhausted melanoma-reactive cells in peripheral blood. These data will inform further development of cancer immunotherapy.Caushi JX, …, Smith KN. Nature 2021 Aug 5;596:126–32.Oliveira G, …, Wu CJ. Nature 2021 Aug 5;596:119–25.A targeted radionuclide and checkpoint inhibition combination enhances antitumor immunity (by MichaelFrey via Wikimedia Commons)External beam radiotherapy enhances responses to immune checkpoint blockade (ICB) in preclinical models. Patel et al. show that a targeted radionuclide therapy comprising the alkylphosphocholine analogue NM600 chelated to yittrium-90 (90Y) enhances responses to ICB in mouse models of melanoma, neuroblastoma, and breast cancer without causing toxicity to normal tissues. The 90Y-NM600 plus ICB combination increases immune-cell infiltration into the tumor, and production of proinflammatory cytokines and clonal expansion of CD8+ T cells in the tumor, highlighting the immunologic mechanisms behind this potential new therapeutic combination.Patel RB, …, Morris ZS. Sci Transl Med 2021 Jul 14;13:eabb3631.TFR cells can suppress antitumor responses induced by ICB (from Fig. 1C of Rodriguez and Engelhard, Cancer Immunol Res 2020)The mechanisms behind the effects of immune checkpoint blockade (ICB) are not completely understood. Eschweiler et al. show that follicular regulatory T (TFR) cells are present in tumor-associated tertiary lymphoid structures (TLS) and that they are more suppressive than regulatory T cells. Anti-PD1 can increase the number of TFR cells in tumors, and administration of anti-CTLA4 to deplete regulatory cells prior to anti-PD1 improves treatment efficacy in mice and humans.Eschweiler S, …, Vijayanand P. Nat Immunol 2021 Aug 1;22:1052–63.β2M can accumulate in the tumor microenvironment (β2M staining, from Fig. 1A of Roemer et al., Cancer Immunol Res 2016)β2-microglobulin (β2M) concentrations increase during progression of multiple myeloma (MM), but β2M's role in disease is not well-known. Hofbauer et al. find that MM-associated macrophages can phagocytose β2M, which then leads to NLRP3-mediated inflammasome activation. The resulting cytokine secretion and inflammation contribute to MM severity. Inhibition of the inflammasome or related cytokines ameliorates disease in a mouse model of MM, thus highlighting potential therapeutic targets for the treatment of MM.Hofbauer D, …, Bruns H. Immunity 2021 Jul 20. DOI: 10.1016/j.immuni.2021.07.002.

Leveraging the Treg-intrinsic CTLA4–PKCη signaling pathway for cancer immunotherapy

BackgroundOur previous studies revealed a critical role of a novel CTLA4-protein kinase C-eta (PKCη) signaling axis in mediating the suppressive activity of regulatory T cells (Tregs) in antitumor immunity. These...

Albumin Fusion at the N-Terminus or C-Terminus of HM-3 Leads to Improved Pharmacokinetics and Bioactivities.

HM-3, an integrin antagonist, exhibits anti-tumor biological responses and therefore has potential as a therapeutic polypeptide. However, the clinical applications of HM-3 are limited by its short half-life. In this study, we genetically fused human serum albumin (HSA) to the N or C-terminus of HM-3 to improve HM-3 pharmacokinetics. HM-3/HSA proteins were successfully expressed in Pichia pastoris and displayed improved pharmacokinetic properties and stability. Among them, the half-life of HM-3-HSA was longer than HSA-HM-3. In vitro, the IC50 values of HSA-HM-3 and HM-3-HSA were 0.38 ± 0.14 μM and 0.25 ± 0.08 μM in B16F10 cells, respectively. In vivo, the inhibition rates of B16F10 tumor growth were 36% (HSA-HM-3) and 56% (HM-3-HSA), respectively, indicating antitumor activity of HM-3-HSA was higher than HSA-HM-3. In conclusion, these results suggested that the HM-3/HSA fusion protein might be potential candidate HM-3 agent for treatment of melanoma and when HSA was fused at the C-terminus of HM-3, the fusion protein had a higher stability and activity.