B16F10 Tumor Research Articles

Effects of Electrochemotherapy on Immunologically Important Modifications in Tumor Cells.

Electrochemotherapy (ECT) is a clinically acknowledged method that combines the use of anticancer drugs and electrical pulses. Electrochemotherapy with bleomycin (BLM) can induce immunogenic cell death (ICD) in certain settings. However, whether this is ubiquitous over different cancer types and for other clinically relevant chemotherapeutics used with electrochemotherapy is unknown. Here, we evaluated in vitro in the B16-F10, 4T1 and CT26 murine tumor cell lines, the electrochemotherapy triggered changes in the ICD-associated damage-associated molecular patterns (DAMPs): Calreticulin (CRT), ATP, High Mobility Group Box 1 (HMGB1), and four immunologically important cellular markers: MHCI, MHC II, PD-L1 and CD40. The changes in these markers were investigated in time up to 48 h after ECT. We showed that electrochemotherapy with all three tested chemotherapeutics induced ICD-associated DAMPs, but the induced DAMP signature was cell line and chemotherapeutic concentration specific. Similarly, electrochemotherapy with CDDP, OXA or BLM modified the expression of MHC I, MHC II, PD-L1 and CD40. The potential of electrochemotherapy to change their expression was also cell line and chemotherapeutic concentration specific. Our results thus put the electrochemotherapy with clinically relevant chemotherapeutics CDDP, OXA and BLM on the map of ICD inducing therapies.

Targeting the tumor microenvironment by liposomal Epacadostat in combination with liposomal gp100 vaccine

Indoleamine-2,3-dioxygenase (IDO1) pathway has vital role in cancer immune escape and its upregulation leads to immunosuppressive environment which is associated with poor prognosis and progression in various cancers like melanoma. Previously, we showed the antitumoral efficacy of nanoliposomal form of Epacadostat (Lip-EPA), as an IDO1 inhibitor. Herein, we used Lip-EPA as a combination approach with liposomal gp100 (Lip-gp100) anti-cancer vaccine in melanoma model. Here, we showed that B16F10 tumor express IDO1 so using Lip-EPA will enhance the efficacy of vaccine therapy. The biodistribution of ICG-labelled liposomal form of EPA showed the remarkable accumulation of drug at tumor site. In an in vivo study, Lip-EPA enhanced the antitumor efficacy of Lip-gp100 in which the IDO mRNA expression was decreased (~ fourfold) in tumor samples. Also, we identified a significant increase in the number of infiltrated T lymphocytes (p < 0.0001) with enhanced in interferon gamma (IFN-γ) production (p < 0.0001). Additionally, Lip-EPA + Lip-gp100 significantly modulated intratumoral regulatory T cells which altogether resulted in the highest delay in tumor growth (TGD = 56.54%) and increased life span (ILS > 47.36%) in treated mice. Our study demonstrated that novel combination of Lip-EPA and Lip-gp100 was effective treatment with capability of being used in further clinical studies.

Abstract 6408: Anti-tumor immunity evolved by novel titanium peroxide nanoparticles (TiOxNPs) as a radiosensitizer and PD-1 blockade

Abstract Abstract: Release and presentation of tumor antigens are the preconditions of cellular immune response. Radiotherapy have been reported to release endogenous tumor antigens and enhanced the adjuvanticity from dying cancer cells via endoplasmic reticulum (ER) stress, known as immunogenic cell death. We developed a novel radiotherapy using radiosensitizer (TiOxNPs) that could drive effective anti-tumor responses. The TiOxNPs were robustly radiation-induced reactive oxygen species (ROS) generation. This burst therapy could enhance to release and presentation of endogenous tumor antigens, subsequently cross-presentation of antigens by Dendritic cells (DCs) for T-cell activation in secondary lymphoid organs (SLO). Methods: B16-F10 murine melanoma cells (5 × 105) were subcutaneously injected into the right flank of C57BL/6J respectively. Mice were divided into five treatment groups: 1) untreated; 2) XRT; 3) XRT+ TiOxNPs; 4) XRT+anti-PD-1Ab; and 5) XRT+ TiOxNPs +anti-PD-1Ab. B16-F10 tumors were intratumorally injected with TiOxNPs (8mg/kg) on days 10 and received 5Gy or 10Gy X-ray radiation approximately 1 hour after the injection. Anti-PD-1 antibody (200µg) intraperitoneally administered to the mice on the first day of X-ray irradiation, days 13 and 16. The tumor volume were assessed every 2-3 days after treatment. Mice were sacrificed after days 17 and isolated spleen and tumor. Results: TiOxNPs acted as a potent radiosensitizer, which enhanced cytotoxic ROS-mediated cell death. This combination effect could not only a local treatment that directly kills cancer cells but also could enhance the adjuvanticity from dying cancer cells by DAMPs, and significantly upregulate expression of MHC class I on tumor cells. Treatment of B16-F10 melanoma tumor model with burst therapy resulted in tumor shrinkage and extended overall survival. Burst therapy facilitated the recruitment and maturation of DCs into SLO, and improved antigen presentation to naive T cells. Infiltration into the tumor site of CTL were correlated with increasing of migration DCs into SLO. Conclusion: A novel radiotherapy combining with TiOxNPs played an important role in release of endogenous tumor antigens and DAMPs. The upregulation of MHC class I expression on tumor cells after X-ray irradiation facilitated the recognition of the tumor cells by T cells, leading to significantly enhanced antitumor immune response compared to exisiting immunoradiotherapy. This novel nanoparticles exposed to X-ray irradiation could trigger of antigen-specific immune responses and transform a tumor into a site for activation of antitumor immune response. Citation Format: Yoshiko Fujita, Hikaru Kubota, Chiaki Ogino, Ryohei Sasaki. Anti-tumor immunity evolved by novel titanium peroxide nanoparticles (TiOxNPs) as a radiosensitizer and PD-1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6408.

Abstract 3580: Noninvasive immuno-PET imaging of CD8+T cell behavior in tumor bearing mice models treated with SAR444245

Abstract SAR444245 (formerly THOR-707) is a site-specific pegylated recombinant human IL-2 molecule which blocks IL-2R alpha-binding while retains near-native affinity for beta/gamma IL-2 receptor subunits. SAR444245 reduces B16-F10 tumor proliferation in C57BL/6 mice and induces a short transient migration of CD8+ T and NK cells from peripheral blood to lymphoid organs before a strong proliferation (Ptacin et al, Nat Commun 2021). Here we report CD8+ T cells imaging post SAR444245 as monotherapy in naive and tumor bearing mice using nuclear imaging. An anti-murine CD8+ PET probe was injected in mice 24 hours post SAR444245 administration and nuclear imaging was performed at different times over 6 days. In C57BL/6 naive and in poorly immunogenic B16-F10 tumor bearing animals, images reveal a decreased Standardized Uptake Value (SUV) within blood in treated animals at 3mg/kg compared to control ones. In parallel, blood SUV Ratio (SUVR) increases within spleen, lymph nodes and thymus after SAR444245 injection compared to control mice. On tumor site, a stronger SUVR is measured at 144 hours post injection in SAR444245 treated group than in the control group. A dose escalation ranging from 1 to 6 mg/kg of SAR444245 was performed in highly immunogenic CT26 tumor bearing animal. Blood SUV decreases in the treated groups compared to the control group. In lymphoid organs, all SUVR increase at 144 hours post injection in the treated groups compared to the control one. In CT26 tumors, a slight increase of the SUVR is observed for groups treated at 3 and 6 mg/kg. In parallel, a pronounced reduction of tumor growth is observed in the treated group at 6mg/kg. These studies non-invasively confirm the mode of action of SAR444245 with a rapid relocation of CD8+ T cells from peripheral blood to lymphoid organs and tumor site. Citation Format: Sebastion d'Heilly, Fabrice Tirode, Maxime Gaulin, Dudzicki Anne, Guillaume Bluet, Stéphane Guerif, Francis Descamps, Peter Casteels, Xiangming Li, Rui Wang, Robin Meng, Erwan Jouannot. Noninvasive immuno-PET imaging of CD8+T cell behavior in tumor bearing mice models treated with SAR444245. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3580.

Abstract 1135: TP317, a first-in-class resolvin E1 small molecule, potentiates the efficacy of immune checkpoint (ICI) inhibitors in ICI-resistant and ICI-sensitive tumors

Abstract Background: TP317 is a novel, highly stable chelate salt of resolvin E1 (RvE1), a pro-resolution mediator that stimulates myeloid cell phagocytosis of tumor debris and attenuates pro-tumoral inflammation through activation of the GPCR, ChemR23 (Sulciner et al., 2018, J. Exp. Med). We hypothesized that RvE1 shifts the immunosuppressive tumor microenvironment (TME) to an immunogenic state, thus offering combination potential with immune checkpoint inhibitors (ICI) in ICI-resistant and ICI-sensitive tumors. Methods: Subcutaneous murine models of lung (LLC), melanoma (B16F10), and pancreatic (Panc02; KPC) tumors were used to investigate TP-317 monotherapy and combinations with ICI. Treatment was initiated when tumors reached 125-240 mm3. Results: In the LLC model (N=5/group), TP317 (0.75 µg QD) inhibited tumor growth (1419 ± 266 mm3) compared to placebo (2348 ± 542 mm3; p=0.068) and anti-PD1 (200 µg IP, Q3D; 3015 ± 730; p=0.078). TP317 + anti-PD1 dual therapy was superior to placebo and anti-PD1 alone (893 ± 166 mm3; p&amp;lt;0.05). In the B16F10 model (N=8/group), TP317 (7.5 µg Q6D) was efficacious compared to placebo (787 ± 367 vs 1964 ± 208 mm3; p&amp;lt;0.01) and comparable to the ICI dual therapy of anti-PD1 + anti-CTLA4 (100 µg 1st dose, 200 µg IP Q3D up to 4 doses). Triple therapy with TP317 + anti-PD1 + anti-CTLA4 was superior to ICI dual therapy (340 ± 91 vs 757 ± 260 mm3; p&amp;lt;0.05). In the Panc02 model (N=8/group), TP317 (0.75 µg Q7D) was efficacious compared to placebo (1148 ± 178 vs 1992 ±165 mm3; p&amp;lt;0.001) and comparable to anti-PD1. TP317 + anti-PD1 dual therapy demonstrated significant anti-tumor activity compared to anti-PD1 alone (329 ± 72 vs 1446 ± 305 mm3; p&amp;lt;0.01). In KRAS-mutant KPC tumors (N=10/group), TP317 (7.5 µg Q6D) demonstrated significant anti-tumor activity compared to placebo (500 ± 110 vs 1314 ± 106 mm3; p&amp;lt;0.001) and was comparable to anti-PD1, while TP-317 + anti-PD1 was superior to anti-PD1 alone (353 ± 82 vs 710 ± 106 mm3; p&amp;lt;0.01). In Panc02 and KPC models, TP317’s anti-tumor efficacy was attenuated by CD8+ T cell or NK cell depletion. Consistent with the depletion study results, RNAseq analysis with cell-type deconvolution showed that TP317 enhanced CD8 and NK cell associated programs in Panc02 and B16F10 tumors, and also promoted macrophage, dendritic cell, B cell and antigen presentation functions in the TME. Conclusions: TP317 monotherapy and ICI combinations significantly inhibited tumor growth in various murine models of cancer. The effects on various immune cell functions and the surprising efficacy of short half-life RvE1 (&amp;lt;2 hours) dosed weekly suggests that TP317 is reprogramming the TME to an immunogenic state. In summary, TP317, an RvE1 drug with a high therapeutic index, has potent single agent efficacy and offers a novel approach in combination with ICI to treat ICI-resistant and ICI-sensitive tumors. Citation Format: Franciele C. Kipper, Eva Rothenberger, Abigail Kelly, Michael Gillespie, Ahmed Attaya, Diane R. Bielenberg, Sui Huang, Lance Pflieger, Frank Sciavolino, Aaron Mathias, Wayne Klohs, John Parkinson, Gary Mathias, Dipak Panigrahy. TP317, a first-in-class resolvin E1 small molecule, potentiates the efficacy of immune checkpoint (ICI) inhibitors in ICI-resistant and ICI-sensitive tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1135.

Abstract 4127: ANV600 is a potent, Cis-signaling, non-alpha IL-2 agonist which efficiently expands intratumoral stem-like CD8 T cells

Abstract Non-alpha IL-2-based therapeutic modalities with preferential signaling through the IL-2 beta(CD122)/gamma(CD132) receptor are in clinical development and have the potential to substantially increase the therapeutic index of recombinant IL-2 (aldesleukin) for cancer therapy. ANV600 is a novel bispecific compound, which features an anti-IL-2 antibody/IL-2 fusion protein and a proprietary hPD-1 binding moiety for specific delivery of the non-alpha IL-2 to tumor antigen experienced PD-1+ T cells. Compared to the untargeted control, ANV600 has increased potency to induce STAT5 phosphorylation in human PD-1+CD8 T cells in vitro. At the same time, stimulation with ANV600 results in markedly reduced STAT5 phosphorylation on Treg cells compared to aldesleukin. In activated human PBMCs, ANV600 induces PD-1 co-internalization with the CD122-CD132 complex, reducing detectable PD-1 on CD8 and CD4 T-cells. Surface PD-1 levels decrease is not observed on cells incubated with untargeted bispecific compound. In transgenic human PD-1 mice ANV600 treatment leads to marked tumor growth retardation in the B16F10 and MC38 subcutaneous tumor models compared to untargeted compound and vehicle. Immunophenotyping of the tumor infiltrating lymphocytes revealed a dose-dependent increase of intratumoral stem-like PD-1+T cells and cytotoxic GrzB+PD-1+T cells in mice treated with ANV600. ANV600 has potent and selective effects on antigen-experienced CD8 T cells both in human PBMCs and mouse syngeneic tumor models. This agent may be a promising anti-tumor therapeutic against poorly immunogenic tumors and warrants further pharmaceutical development. Citation Format: Patrizia Murer, Ulisse Salazar, Nicole Egli, Laetitia Petersen, Pia Neubert, Kirsten Richter, Christian Stocker, Alexander Rau, Andreas Katopodis, Christoph Huber. ANV600 is a potent, Cis-signaling, non-alpha IL-2 agonist which efficiently expands intratumoral stem-like CD8 T cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4127.

Abstract 4499: The peptide-drug conjugate sudocetaxel zendusortide (TH1902) potentiates anti-tumoral activity of the anti-PD-L1 checkpoint inhibitor and induces immune cell infiltration in a B16-F10 syngeneic melanoma model

Abstract Tumor-infiltrating immune cells are involved in the control of cancer and are closely related to clinical outcomes. Sudocetaxel Zendusortide (TH1902), a peptide-drug conjugate (PDC) of the sortilin (SORT1)-binding peptide TH19P01 ester-linked to two docetaxel moieties, has been shown to exert superior anti-cancer activities in multiple cancer models including melanoma syngeneic and xenograft murine models. Melanomas express elevated levels of SORT1 and are considered as one of the most immunogenic tumors where immune checkpoint inhibitors are among the standard of care treatment used. Here, we first questioned whether TH1902 anti-cancer effects involved infiltration of immune cells in a murine SORT1-positive syngeneic, non-immunogenic and highly aggressive B16-F10 melanoma model in C57BL/6 mice. Weekly administration of docetaxel reduced by half the growth of B16-F10 tumor allografts, while administration of TH1902 as a single agent and at equivalent docetaxel doses, was well tolerated and resulted in tumor regression after only 2 treatments. Surprisingly, in this immunologically cold tumor, immunohistochemistry analysis of tumors for immune cell infiltration showed a net increase in total leukocytes (CD45+) infiltration within TH1902-treated tumors compared with docetaxel-treated tumors, especially with for tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs). Furthermore, the increased staining intensities for Perforin and Granzyme B were indicative of the elevated cytotoxic T and natural killer (NK) cells activity in TH1902-treated tumors. This corroborated with increased caspase-3 apoptotic activity. TH1902 and docetaxel doses were next halved and combined with the checkpoint inhibitor anti-PD-L1. In a first study, the TH1902/anti-PD-L1 combination resulted in increased tumor growth inhibition when compared with both agents alone. Interestingly, TH1902 as a single agent showed better tumor growth inhibition than docetaxel or docetaxel/anti-PD-L1 combination. In a second study, the TH1902/anti-PD-L1 combination significantly increased animal survival over either anti-PD-L1 or TH1902 as single agents (21 days increased median survival compared to 2.5 and 12.5 days respectively). We conclude that the superiority of TH1902 anticancer activity over docetaxel involves, in part, the modulation of infiltrating immune cells within the tumor microenvironment. This is the first demonstration that immune cell infiltration patterns play a pivotal role in the TH1902-associated anti-tumoral response. Combination of TH1902 with checkpoint inhibitors (anti-PD-L1) further reveals that this may lead to improved clinical outcomes in future immunotherapy translational approaches. Citation Format: Michel Demeule, Jean-Christophe Currie, Cyndia Charfi, Alain Zgheib, Isabelle Cousineau, Richard Béliveau, Christian Marsolais, Borhane Annabi. The peptide-drug conjugate sudocetaxel zendusortide (TH1902) potentiates anti-tumoral activity of the anti-PD-L1 checkpoint inhibitor and induces immune cell infiltration in a B16-F10 syngeneic melanoma model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4499.

Abstract 2282: Immune modulation of the T cell repertoire in mice following immunotherapy and chemotherapy combination treatments

Abstract Background: Immune checkpoint blockade therapeutics have revolutionized cancer therapy. However, most patients are either unresponsive or develop acquired resistance. Understanding patient-specific temporal T cell repertoires in the context of immune interventions will contribute to a better understanding of potential improvement strategies for better T cell anti-tumor immunity. Current methods to understand the T cell repertoire changes over time are limited due to the limited amount of material and prohibitive cost. In this study, we designed and performed longitudinal TCR sequencing in mice treated with multiple therapies. Methods: C57BL/6 mice were used in each treatment group, with blood drawn overtime before, during and after treatment. Mice were treated with a combination of immunotherapeutic agents and OVA peptide immunization, 100µl blood was drawn at multiple pre-, on- and post-treatment time points followed subsequently by TCR sequencing. Cyclophosphamide (CTX) is known to cause lymphodepletion followed by homeostatic proliferation. Thus, we also treated B16-F10 tumor bearing mice with CTX, and performed TCR sequencing on blood draws pre-, on-, and post-treatment to track TCR clone diversity and clonality over time. Results: We find that our method is sensitive enough to detect effects of immunotherapies on TCR diversity in small sample quantities. Using our method, we discovered that certain immunotherapeutic agents, most evidently CD40, have a significant but transient effect on the diversity of the T cell repertoire. We observed an initial increase in repertoire clonality, seven days post CD40 treatment followed by contraction. We show how we can identify antigen specific T cells and follow reacting T cell clones. In addition, we find that CTX treatment is associated with increased clonality in tumor bearing mice particularly in the week following treatment reflecting changes in the TCR repertoire. Conclusion: We have developed a method by which we can follow the TCR repertoire in mice treated with immunotherapy or chemotherapy. This enabled us to isolate and study the effects of different immune modulatory drugs of the endogenous T cell repertoire overtime. Citation Format: Yuval Elhanati, Mariam M. George, Hyejin Choi, Rachana Maniyar, Linda Hamadane, Neeman Mohibullah, Benjamin D. Greenbaum, Taha Merghoub. Immune modulation of the T cell repertoire in mice following immunotherapy and chemotherapy combination treatments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2282.

Potential anti-cancer effects of hibernating common carp (Cyprinus carpio) plasma on B16-F10 murine melanoma: In vitro and in vivo studies

Melanoma is the major type of skin cancer, which its treatment is still a challenge in the world. In recent years, interest in hibernation-based therapeutic approaches for various biomedical applications has been increased. Many studies indicated that some factors in the blood plasma of hibernating animals such as alpha-2-macroglobulin (A2M) cause anti-proliferative effects. Considering that, the present study was conducted to investigate the anti-cancer effects of hibernating common carp plasma (HCCP) on murine melanoma (B16-F10) in vitro and in vivo. The effect of HCCP on cell viability, migration, apoptosis rate, and cell cycle distribution of B16-F10 cells, tumor growth, and rate of survival were evaluated. To investigate the role of A2M in the anti-cancer effects of HCCP, the gene of interest and proteins in HCCP and non-hibernating common carp plasma (NHCCP) were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry analysis. Based on our findings, HCCP significantly decreased B16-F10 cell viability. Moreover, HCCP caused morphological alternations, inhibition of migration, induction of apoptosis, and significantly induced the cell cycle arrest at the G2/M phase. In addition, A2M level was significantly increased in HCCP compared with NHCCP. Taken together, our findings suggested that HCCP had the potential to be a promising novel therapeutic target for cancer treatment because of its anti-cancer properties.

Nanoparticles that Transcytosed through Cancer Cells Can Elicit Immune Response.

Transcytosis is a crucial process that nanomedicines can experience in various delivery stages. However, little was known about whether it endows biofunctions to the nanomedicines. Here, we reported that transporting photodynamic nanoparticles across cancer cells via the endoplasmic reticulum (ER)-Golgi pathway formulated them with abundant neoantigens and damage-associated molecular patterns. The resultant nanoparticles (Tran-NPs) were potent in dendritic cell maturation and T cell activation. Meanwhile, the photodynamic Tran-NPs maintained their primary function of repolarizing immunosuppressive cells. The immune responses were observed in melanoma B16F10 tumor models. Our work suggested that the transcytosis process reprogrammed the nanoparticles with immunological properties, which might shed light on the design of nanomedicines.

AAZTA-Like Ligands Bearing Phenolate Arms as Efficient Chelators for 68 Ga Labelling in vitro and in vivo.

The introduction of a phenolate pendant arm in place of an acetate on AAZTA- and DATA-like ligands resulted in hepta- and hexadentate chelators able to form Ga(III) complexes with thermodynamic stability and kinetic inertness higher than that of other Ga(III) complexes based on the parent 6-amino-6-methylperhydro-1,4-diazepine scaffold. In particular, the heptadentate AAZ3A-endoHB with a phenolate arm on an endocyclic N-atom shows a logKGaL of 27.35 and a remarkable resistance to hydroxide coordination up to basic pH (pH>9). This behaviour allows to also improve the kinetic inertness of the complex showing a dissociation half-life (t1/2 ) at pH 7.4 of 76 h. Although also the hexadentate AAZ2A-exoHB chelator forms a stable (logKGaL =24.69) and inert (t1/2 =33 h at pH 7.4) Ga(III) complex, the 68 Ga labelling showed a better radiochemical yield with AAZ3A-endoHB, especially at room temperature. Thus, a bifunctional chelator of AAZ3A-endoHB was synthesized bearing an isothiocyanate group that was conjugated to the N-terminus of a c(RGD) peptide for integrin receptor targeting. Finally, the conjugate was successfully labelled with 68 Ga isotope, and the resulting radiotracer tested for its stability in human serum and then in vivo for targeting B16-F10 tumours with miniPET imaging.

CaCO3 based proton nanosponge to potentiate immune checkpoint blockade therapy by synergistically reversing tumor immunosuppression

Tumor acidity presents one of the leading causes of tumor immunosuppression (e.g., exhaustion of effector T cells), thereby remarkably attenuating the potency of current immune checkpoint blockade (ICB) therapy in controlling tumor progression. Here, we report that calcium carbonate encapsulated with copolymers of poly(lactic-co-glycolic acid) (PLGA) and PLGA-poly(ethylene glycol) (PLGA-PEG) can work as a proton nanosponge to neutralize tumor acidity by reacting with protons post intravenous administration. The obtained CaCO3@PLGA NPs can thus promote the reinvigoration of both exhausted CD8+ T cells and innate antitumor immunity. As a result, CaCO3@PLGA NP administration could potentiate the therapeutic efficacies of three ICB therapies toward both CT26 and B16F10 tumor xenografts by preventing exhaustion of CD8+ T cells and activating innate antitumor immunity. Furthermore, we further demonstrate that intratracheal administration of CaCO3@PLGA NPs effectively suppressed lung metastasis of B16F10 melanoma when synergized with systemic anti-PD-1 administration. This work highlights that CaCO3 nanoparticles could be an effective yet safe nanomedicine to generally reinforce ICB therapies toward diverse types of tumors by reversing tumor immunosuppression, promising for future clinical translation.

Short-chain fatty acid-releasing nano-prodrugs for attenuating growth and metastasis of melanoma.

Low-molecular-weight (LMW) short-chain fatty acids (SCFAs), such as propionic and butyric acids, have been reported to possess anti-neoplastic effects; however, rapid renal clearance and high dose-based side effects limit their clinical translation. Hence, in this study, we have designed a new self-assembling nano-prodrugs that can effectively supply SCFAs: endogenous enzyme-metabolizable block copolymer poly(ethylene glycol)block-poly(vinyl ester) possessing several units of SCFAs conjugated as side chains via ester linkages. These amphiphilic polymers spontaneously self-assemble into nanostructures under aqueous conditions to form orally administrable nano-prodrugs (butyric acid: NanoBA and propionic acid: NanoPA). Herein, we show the therapeutic efficacy of SCFA nanoparticles (NanoSCFA) in a mouse model of metastasis (melanoma). Ad libitum intake of our NanoSCFA markedly demonstrated a decrease in the metastatic tumor nodules in the lungs compared with the effect observed after LMW SCFA administration with no discernible toxicity to the GI tract. In contrast, LMW SCFAs, even at a lower concentration than that of the NanoSCFA, facilitated villus atrophy. Taken together, our work suggests that the use of NanoSCFA as a therapeutic intervention for metastatic cancer is preferable over typical LMW SCFAs. STATEMENT OF SIGNIFICANCE: Low-molecular-weight (LMW) short-chain fatty acids (SCFAs) have shown versatile therapeutic effects on various diseases, including anti-tumorigenesis effects. However, their clinical translation is limited due to their poor pharmacokinetic profile and adverse effects. To overcome these limitations, we have developed new amphiphilic block copolymer-based SCFA-prodrugs, which self-assemble into nanoparticles in aqueous media (NanoSCFA). SCFAs are covalently conjugated to the hydrophobic polymer segment via ester linkage, which can be enzymatically metabolized after oral administration. In the present study, we confirmed that ad libitum intake of NanoSCFAs retarded the growth and metastatic potential of B16-F10 tumors compared to the LMW SCFAs with negligible discernible toxicity, reflecting NanoSCFA as a preferable therapeutic intervention to LMW SCFA counterparts.

Murine regulatory T cells utilize granzyme B to promote tumor metastasis.

Regulatory T cells (Tregs) possess a wide range of mechanisms for immune suppression. Among them, Granzyme B (GzmB) and perforin expressed by Tregs were shown to inhibit tumor clearance in previous reports, which contradicted the canonical roles of these cytotoxic molecules expressed by cytotoxic T cells and NK cells in antitumor immune responses. Given the ability of the tumor to manipulate the microenvironment, Treg-derived GzmB function may represent an important approach to aid in tumor growth as well as facilitating tumor metastasis. In this study, we utilized Treg-specific GzmB knockout (Foxp3creGzmBfl/fl) mice to test whether Treg-derived GzmB can aid in tumor progression and metastasis. Using an IL-2 complex to activate GzmB expression in the non-immunogenic B16-F10 tumor model, we provide evidence to show that GzmB produced by Tregs is important for spontaneous metastasis to the lungs. In addition, we depleted CD8 + T cells to selectively measure the impact of Treg-derived GzmB in an experimental lung metastasis model by intravenous injection of B16-F10 tumor cells; our results demonstrate that Treg-derived GzmB plays an important role in increasing the metastatic burden to the lungs.

Identification of CBPA as a New Inhibitor of PD-1/PD-L1 Interaction.

Targeting of the PD-1/PD-L1 immunologic checkpoint is believed to have provided a real breakthrough in the field of cancer therapy in recent years. Due to the intrinsic limitations of antibodies, the discovery of small-molecule inhibitors blocking PD-1/PD-L1 interaction has gradually opened valuable new avenues in the past decades. In an effort to discover new PD-L1 small molecular inhibitors, we carried out a structure-based virtual screening strategy to rapidly identify the candidate compounds. Ultimately, CBPA was identified as a PD-L1 inhibitor with a KD value at the micromolar level. It exhibited effective PD-1/PD-L1 blocking activity and T-cell-reinvigoration potency in cell-based assays. CBPA could dose-dependently elevate secretion levels of IFN-γ and TNF-α in primary CD4+ T cells in vitro. Notably, CBPA exhibited significant in vivo antitumor efficacy in two different mouse tumor models (a MC38 colon adenocarcinoma model and a melanoma B16F10 tumor model) without the induction of observable liver or renal toxicity. Moreover, analyses of the CBPA-treated mice further showed remarkably increased levels of tumor-infiltrating CD4+ and CD8+ T cells and cytokine secretion in the tumor microenvironment. A molecular docking study suggested that CBPA embedded relatively well into the hydrophobic cleft formed by dimeric PD-L1, occluding the PD-1 interaction surface of PD-L1. This study suggests that CBPA could work as a hit compound for the further design of potent inhibitors targeting the PD-1/PD-L1 pathway in cancer immunotherapy.

Lactate oxidase/vSIRPα conjugates efficiently consume tumor-produced lactates and locally produce tumor-necrotic H2O2 to suppress tumor growth

Aggressive tumor formation often leads to excessive anaerobic glycolysis and massive production and accumulation of lactate in the tumor microenvironment (TME). To significantly curb lactate accumulation in TME, in this study, lactate oxidase (LOX) was used as a potential therapeutic enzyme and signal regulatory protein α variant (vSIRPα) as a tumor cell targeting ligand. SpyCatcher protein and SpyTag peptide were genetically fused to LOX and vSIRPα, respectively, to form SC-LOX and ST-vSIRPα and tumor-targeting LOX/vSIRPα conjugates were constructed via a SpyCatcher/SpyTag protein ligation system. LOX/vSIRPα conjugates selectively bound to the CD47-overexpressing mouse melanoma B16-F10 cells and effectively consumed lactate produced by the B16-F10 cells, generating adequate amounts of hydrogen peroxide (H2O2), which induces drastic necrotic tumor cell death. Local treatments of B16-F10 tumor-bearing mice with LOX/vSIRPα conjugates significantly suppressed B16-F10 tumor growth in vivo without any severe side effects. Tumor-targeting vSIRPα may allow longer retention of LOX in tumor sites, effectively consuming surrounding lactate in TME and locally generating adequate amounts of cytotoxic H2O2 to suppress tumor growth. The approach restraining the local lactate concentration and H2O2 in TME using LOX and vSIRPα could offer new opportunities for developing enzyme/targeting ligand conjugate-based therapeutic tools for tumor treatment.

Targeted Degradation of PD-L1 and Activation of the STING Pathway by Carbon-Dot-Based PROTACs for Cancer Immunotherapy.

Proteolysis targeting chimeras (PROTACs) technology is an emerging approach to degrade disease-associated proteins. Here, we report carbon-dot (CD)-based PROTACs (CDTACs) that degrade membrane proteins via the ubiquitin-proteasome system. CDTACs can bind to programmed cell death ligand 1 (PD-L1), recruit cereblon (CRBN) to induce PD-L1 ubiquitination, and degrade them with proteasomes. Fasting-mimicking diet (FMD) is also used to enhance the cellular uptake and proteasome activity. More than 99 % or 90 % of PD-L1 in CT26 or B16-F10 tumor cells can be degraded by CDTACs, respectively. Furthermore, CDTACs can activate the stimulator of interferon genes (STING) pathway to trigger immune responses. Thus, CDTACs with FMD treatment effectively inhibit the growth of CT26 and B16-F10 tumors. Compared with small-molecule-based PROTACs, CDTACs offer several advantages, such as efficient membrane protein degradation, targeted tumor accumulation, immune system activation, and in vivo detection.

A tactical nanomissile mobilizing antitumor immunity enables neoadjuvant chemo-immunotherapy to minimize postsurgical tumor metastasis and recurrence.

Neoadjuvant chemotherapy has become an indispensable weapon against high-risk resectable cancers, which benefits from tumor downstaging. However, the utility of chemotherapeutics alone as a neoadjuvant agent is incapable of generating durable therapeutic benefits to prevent postsurgical tumor metastasis and recurrence. Herein, a tactical nanomissile (TALE), equipped with a guidance system (PD-L1 monoclonal antibody), ammunition (mitoxantrone, Mit), and projectile bodies (tertiary amines modified azobenzene derivatives), is designed as a neoadjuvant chemo-immunotherapy setting, which aims at targeting tumor cells, and fast-releasing Mit owing to the intracellular azoreductase, thereby inducing immunogenic tumor cells death, and forming an in situ tumor vaccine containing damage-associated molecular patterns and multiple tumor antigen epitopes to mobilize the immune system. The formed in situ tumor vaccine can recruit and activate antigen-presenting cells, and ultimately increase the infiltration of CD8+ T cells while reversing the immunosuppression microenvironment. Moreover, this approach provokes a robust systemic immune response and immunological memory, as evidenced by preventing 83.3% of mice from postsurgical metastasis or recurrence in the B16-F10 tumor mouse model. Collectively, our results highlight the potential of TALE as a neoadjuvant chemo-immunotherapy paradigm that can not only debulk tumors but generate a long-term immunosurveillance to maximize the durable benefits of neoadjuvant chemotherapy.

SARS-CoV-2 RBD protein enhances the oncolytic activity of the vesicular stomatitis virus.

Despite recent advances in the research on oncolytic viruses (OVs), a better understanding of how to enhance their replication is key to improving their therapeutic index. Understanding viral replication is important to improve treatment outcomes based on enhanced viral spreading within the tumor milieu. The VSV-Δ51 oncolytic virus has been widely used as an anticancer agent with a high selectivity profile. In this study, we examined the role of the SARS-CoV-2 spike protein receptor-binding domain (RBD) in enhancing VSV-Δ51 viral production and oncolytic activity. To test this hypothesis, we first generated a novel VSV-Δ51 mutant that encoded the SARS-COV-2 RBD and compared viral spreading and viral yield between VSV-Δ51-RBD and VSV-Δ51 in vitro. Using the viral plaque assay, we demonstrated that the presence of the SARS-CoV-2 RBD in the VSV-Δ51 genome is associated with a significantly larger viral plaque surface area and significantly higher virus titers. Subsequently, using an ATP release-based assay, we demonstrated that the SARS-CoV-2 RBD could enhance VSV-Δ51 oncolytic activity in vitro. This observation was further supported using the B16F10 tumor model. These findings highlighted a novel use of the SARS-CoV-2 RBD as an anticancer agent.

Targeting Redundant ROBO1 and SDF-1 Pathways Prevents Adult Hemangioblast Derived-EPC and CEC Activity Effectively Blocking Tumor Neovascularization.

Neovascularization is a key therapeutic target for cancer treatment. However, anti-angiogenic therapies have shown modest success, as tumors develop rapid resistance to treatment owing to activation of redundant pathways that aid vascularization. We hypothesized that simultaneously targeting different pathways of neovascularization will circumvent the current issue of drug resistance and offer enhanced therapeutic benefits. To test this hypothesis, we made use of two distinct models of tumor-neovascularization, which exhibit equally dense microvasculature but show disparate sensitivity to anti-SDF-1 treatment. Lewis lung carcinoma (LLC) is primarily a vasculogenic-tumor that is associated with HSC functioning as a hemangioblast to generate circulating Endothelial Progenitor Cells contributing to formation of new blood vessels, and responds to anti-SDF-1 treatment. B16F0 melanoma is an angiogenic-tumor that derives new blood vessels from existing vasculature and is resistant to anti-SDF-1 therapy. In this study, we observed increased expression of the angiogenic-factor, Robo1 predominantly expressed on the blood vessels of B16F0 tumor. Blockade of Robo1 by the decoy receptor, RoboN, resulted in reduced microvascular-density and tumor-growth. However, this was associated with mobilization of BM-cells into the B16F0 tumor, thus switching the mode of neovascularization from angiogenic to vasculogenic. The use of a combinatorial treatment of RoboN and the monoclonal anti-SDF-1 antibody effectively attenuated tumor-growth and inhibited both angiogenic and BM-derived microvessels.