Sulfoxide-containing polymers conjugated prodrug micelles with enhanced anticancer activity and reduced intestinal toxicity.

Poly(ethylene glycol) (PEG) is widely utilized as a hydrophilic coating to extend the circulation time and improve the tumor accumulation of polymeric micelles. Nonetheless, PEGylated micelles often activate complement proteins, leading to accelerated blood clearance and negatively impacting drug efficacy and safety. Here, we have crafted amphiphilic block copolymers that merge hydrophilic sulfoxide-containing polymers (psulfoxides) with the hydrophobic drug 7-ethyl-10-hydroxylcamptothecin (SN38) into drug-conjugate micelles. Our findings show that the specific variant, PMSEA-PSN38 micelles, remarkably reduce protein fouling, prolong blood circulation, and improve intratumoral accumulation, culminating in significantly increased anti-cancer efficacy compared with PEG-PSN38 counterpart. Additionally, PMSEA-PSN38 micelles effectively inhibit complement activation, mitigate leukocyte uptake, and attenuate hyperactivation of inflammatory cells, diminishing their ability to stimulate tumor metastasis and cause inflammation. As a result, PMSEA-PSN38 micelles show exceptional promise in the realm of anti-metastasis and significantly abate SN38-induced intestinal toxicity. This study underscores the promising role of psulfoxides as viable PEG substitutes in the design of polymeric micelles for efficacious anti-cancer drug delivery.

Neutrophil Extracellular Traps-Inhibiting and Fouling-Resistant Polysulfoxides Potently Prevent Postoperative Adhesion, Tumor Recurrence, and Metastasis.

Peritoneal metastasis (PM) is considered one of the most dreaded forms of cancer metastases for both patients and physicians. Aggressive cytoreductive surgery (CRS) is the primary treatment for peritoneal metastasis. Unfortunately, this intensive treatment frequently causes clinical complications, such as postoperative recurrence, metastasis, and adhesion formation. Emerging evidence suggests that neutrophil extracellular traps (NETs) released by inflammatory neutrophils contribute to these complications. Effective NET-targeting strategies thus show considerable potential in counteracting these complications but remain challenging. Here, one type of sulfoxide-containing homopolymer, PMeSEA, with potent fouling-resistant and NET-inhibiting capabilities, is synthesized and screened. Hydrating sulfoxide groups endow PMeSEA with superior nonfouling ability, significantly inhibiting protein/cell adhesion. Besides, the polysulfoxides can be selectively oxidized by ClO- which is required to stabilize the NETs rather than H2O2, and ClO- scavenging effectively inhibits NETs formation without disturbing redox homeostasis in tumor cells and quiescent neutrophils. As a result, PMeSEA potently prevents postoperative adhesions, significantly suppresses peritoneal metastasis, and shows synergetic antitumor activity with chemotherapeutic 5-Fluorouracil. Moreover, coupling CRS with PMeSEA potently inhibits CRS-induced tumor metastatic relapse and postoperative adhesions. Notably, PMeSEA exhibits low in vivo acute and subacute toxicities, implying significant potential for clinical postoperative adjuvant treatment.

Bolus administration of remimazolam was superior to midazolam for deep sedation in elderly patients undergoing diagnostic bronchoscopy: A randomized, double-blind, controlled trial.

BACKGROUND: To date, there is no standardized practice for the use of pharmacological sedatives during flexible bronchoscopy, particularly for elderly patients. This exploratory study aimed to assess the efficacy and safety of remimazolam at a single induced dose for deep sedation in elderly patients undergoing diagnostic flexible bronchoscopy (DFB), and compare with midazolam, a commonly used sedative. METHODS: A total of 100 elderly patients (age range 65-80 yr; American Society of Anesthesiologists Physical Status I-III) undergoing DFB were randomly allocated into 2 groups according to the sedatives used for induction: the remimazolam group and the midazolam group. Sedation induction was initiated by an intravenous bolus of remimazolam (0.135 mg/kg) or midazolam (0.045 mg/kg), respectively, both groups were combined with a high-dose of alfentanil (18 µg/kg), and supplemented with high-flow nasal cannula (HFNC) oxygen supply at a flow rate of 45 L/min. If the target depth of sedation was not achieved, propofol would be titrated as a rescue. The primary outcome was the success rate of sedation at a single induced dose to achieve target depth (Ramsay sedation score [RSS] = 4) during induction, intraoperative changes in vital signs, postoperative follow-up situation and incidence of post-bronchoscopy adverse events were evaluated as secondary outcomes. RESULTS: The success rate of sedation in the remimazolam group was significantly higher than that in the midazolam group (65.2% vs 39.6%, P = .013), while the incidence of extra sleep within 6 hours after procedure was lower in the remimazolam group as compared to the midazolam group (10.9% vs 31.3%, P = .016). No statistically significant differences were observed between the 2 groups regarding hemodynamic fluctuations, incidence of hypoxemia, and cough response during the procedure, as well as postoperative recall, willingness to undergo reexamination, and other post-bronchoscopy adverse events. CONCLUSIONS: Bolus administration of remimazolam offers advantages over midazolam for deep sedation in elderly patients undergoing DFB, in terms of a higher success rate of sedation and a lower incidence of extra sleep within 6 hours after procedure, though the safety profiles of both groups were favorable.

Protein Kinase STK24 Promotes Tumor Immune Evasion via the AKT-PD-L1 Axis.

Immunotherapy targeting PD-L1 is still ineffective for a wide variety of tumors with high unpredictability. Deploying combined immunotherapy with alternative targeting is practical to overcome this therapeutic resistance. Here, the deficiency of serine-threonine kinase STK24 is observed in tumor cells causing substantial attenuation of tumor growth in murine syngeneic models, a process relying on cytotoxic CD8+ T and NK cells. Mechanistically, STK24 in tumor cells associates with and directly phosphorylates AKT at Thr21, which promotes AKT activation and subsequent PD-L1 induction. Deletion or inhibition of STK24, by contrast, blocks IFN-γ-mediated PD-L1 expression. Various murine models indicate that in vivo silencing of STK24 can significantly enhance the efficacy of the anti-PD-1 blockade strategy. Elevated STK24 levels are observed in patient specimens in multiple tumor types and inversely correlated with intratumoral infiltration of cytotoxic CD8+ T cells and with patient survival. The study collectively identifies STK24 as a critical modulator of antitumor immunity, which engages in AKT and PD-L1/PD-1 signaling and is a promising target for combined immunotherapy.

RNA-based modulation of macrophage-mediated efferocytosis potentiates antitumor immunity in colorectal cancer.

Tumor-associated macrophages play pivotal roles in tumor progression and metastasis. Macrophage-mediated clearance of apoptotic cells (efferocytosis) supports inflammation resolution, contributing to immune evasion in colorectal cancers. To reverse this immunosuppressive process, we propose a readily translatable RNA therapy to selectively inhibit macrophage-mediated efferocytosis in tumor microenvironment. A clinically approved lipid nanoparticle platform (LNP) is employed to encapsulate siRNA for the phagocytic receptor MerTK (siMerTK), enabling selective MerTK inhibition in the diseased organ. Decreased MerTK expression in tumor-associated macrophages results in apoptotic cell accumulation and immune activation in tumor microenvironment, leading to suppressed tumor growth and better survival in both liver and peritoneal metastasis models of colorectal cancers. siMerTK delivery combined with PD-1 blockade further produces enhanced antimetastatic efficacy with reactivated intratumoral immune milieu. Collectively, LNP-based siMerTK delivery combined with immune checkpoint therapy may present a feasible modality for metastatic colorectal cancer therapy.

Killing tumor-associated bacteria with a liposomal antibiotic generates neoantigens that induce anti-tumor immune responses.

Increasing evidence implicates the tumor microbiota as a factor that can influence cancer progression. In patients with colorectal cancer (CRC), we found that pre-resection antibiotics targeting anaerobic bacteria substantially improved disease-free survival by 25.5%. For mouse studies, we designed an antibiotic silver-tinidazole complex encapsulated in liposomes (LipoAgTNZ) to eliminate tumor-associated bacteria in the primary tumor and liver metastases without causing gut microbiome dysbiosis. Mouse CRC models colonized by tumor-promoting bacteria (Fusobacterium nucleatum spp.) or probiotics (Escherichia coli Nissle spp.) responded to LipoAgTNZ therapy, which enabled more than 70% long-term survival in two F. nucleatum-infected CRC models. The antibiotic treatment generated microbial neoantigens that elicited anti-tumor CD8+ T cells. Heterologous and homologous bacterial epitopes contributed to the immunogenicity, priming T cells to recognize both infected and uninfected tumors. Our strategy targets tumor-associated bacteria to elicit anti-tumoral immunity, paving the way for microbiome-immunotherapy interventions.

Gas embolism during surgical hysteroscopy leading to cardiac arrest and refractory hypokalemia: A case report and review of literature.

RATIONALE: One of the catastrophic complications of surgical hysteroscopy is venous gas embolism (VGE), and this event could cause morbidity and in serious cases may even lead to death. However, in cases of VGE accompanied by refractory hypokalemia is rare and can significantly increase the difficulty of treatment and resuscitation. Here, we successfully treated a patient with fatal VGE during surgical hysteroscopy, accompanied by difficult resuscitation with refractory hypokalemia. PATIENT CONCERNS: We report a rare case of sudden cardiac arrest due to VGE during surgical hysteroscopy, followed by difficult resuscitation with refractory hypokalemia. DIAGNOSIS: VGE was diagnosed by a sudden decrease in EtCO2, a loud mill wheel murmur in the thoracic area, and a small number of air bubbles evacuated from the internal jugular catheter. And refractory hypokalemia was diagnosed by serum potassium levels dropping frequently to as low as 2.0 mmol/L within 36 hours of resuscitation after cardiac arrest. INTERVENTIONS: Our vigilant anesthesiologist noticed the early sign of VGE with a sudden drop in EtCO2, and as the cardiac arrest occurred, interventional maneuvers were implemented quickly including termination of the surgical procedure, adjustment of the patient's position, cardiac resuscitation, continuous chest compression, and correction of electrolyte disturbances, particularly refractory hypokalemia during the early stage of resuscitation. OUTCOMES: The patient regained consciousness 4 days after the cardiac arrest and was discharged 1 month later without any neurological deficits. LESSONS: As a relatively simple procedure, surgical hysteroscopy may have catastrophic complications. This case demonstrates the full course of fatal gas embolism and difficult resuscitation during hysteroscopic surgery, and emphasizes the importance of early detection, prompt intervention, and timely correction of electrolyte disturbances, such as refractory hypokalemia.

Macrophage-evading and tumor-specific apoptosis inducing nanoparticles for targeted cancer therapy.

Extended circulation of anticancer nanodrugs in blood stream is essential for their clinical applications. However, administered nanoparticles are rapidly sequestered and cleared by cells of the mononuclear phagocyte system (MPS). In this study, we developed a biomimetic nanosystem that is able to efficiently escape MPS and target tumor tissues. The fabricated nanoparticles (TM-CQ/NPs) were coated with fibroblast cell membrane expressing tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL). Coating with this functionalized membrane reduced the endocytosis of nanoparticles by macrophages, but increased the nanoparticle uptake in tumor cells. Importantly, this membrane coating specifically induced tumor cell apoptosis via the interaction of TRAIL and its cognate death receptors. Meanwhile, the encapsulated chloroquine (CQ) further suppressed the uptake of nanoparticles by macrophages, and synergized with TRAIL to induce tumor cell apoptosis. The vigorous antitumor efficacy in two mice tumor models confirmed our nanosystem was an effective approach to address the MPS challenge for cancer therapy. Together, our TM-CQ/NPs nanosystem provides a feasible approach to precisely target tumor tissues and improve anticancer efficacy.

Liver fibrosis therapy based on biomimetic nanoparticles which deplete activated hepatic stellate cells.

Liver fibrosis is one of the most common liver diseases with substantial morbidity and mortality. However, effective therapy for liver fibrosis is still lacking. Considering the key fibrogenic role of activated hepatic stellate cells (aHSCs), here we reported a strategy to deplete aHSCs by inducing apoptosis as well as quiescence. Therefore, we engineered biomimetic all-trans retinoic acid (ATRA) loaded PLGA nanoparticles (NPs). HSC (LX2 cells) membranes, presenting the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), were coated on the surface of the nanoparticles, while the clinically approved agent ATRA with anti-fibrosis ability was encapsulated in the inner core. The biomimetic coating of TRAIL-expressing HSC membranes does not only provide homologous targeting to HSCs, but also effectively triggers apoptosis of aHSCs. ATRA could induce quiescence of activated fibroblasts. While TM-NPs (i.e. membrane coated NPs without ATRA) and ATRA/NPs (i.e. non-coated NPs loaded with ATRA) only showed the ability to induce apoptosis and decrease the α-SMA expression in aHSCs, respectively, TM-ATRA/NPs induced both apoptosis and quiescence in aHSCs, ultimately leading to improved fibrosis amelioration in both carbon tetrachloride-induced and methionine and choline deficient L-amino acid diet induced liver fibrosis mouse models. We conclude that biomimetic TM-ATRA/NPs may provide a novel strategy for effective antifibrosis therapy.

Impacts of molybdate and ferric chloride on biohythane production through two-stage anaerobic digestion of sulfate-rich hydrolyzed tofu processing residue.

Biohythane production through one-stage anaerobic digestion of sulfate-rich hydrolyzed tofu processing residue has been hampered by high H2S production. Herein, two-stage anaerobic digestion was investigated with the addition of molybdate (MoO42-; 0.24-3.63 g/L) and ferric chloride (FeCl3; 0.025-5.4 g/L) to the dark fermentation stage (DF) to improve biohythane production. DF supplemented with 1.21 g/L MoO42- increased hydrogen yield by 14.6% over the control (68.39 ml/g-VSfed), while FeCl3 had no effect. Furthermore, the maximum methane yields of methanogenic fermentation were 524.8 and 521.6 ml/g-VSfed with 3.63 g/L MoO42- and 0.6 g/L FeCl3 compared to 466.07 ml/g-VSfed of the control. The maximum yields of biohythane and energy were 796.7 ml/g-VSfed and 21.8 MJ/kg-VSfed with 0.6 g/L FeCl3 when the sulfate removal efficiency was 66.7%, and H2S content was limited at 0.08%. Therefore, adding 0.6 g/L FeCl3 is the most beneficial in improving energy recovery and sulfate removal with low H2S content.

Nanoprodrug ratiometrically integrating autophagy inhibitor and genotoxic agent for treatment of triple-negative breast cancer.

Effective combination therapies are urgently needed to treat triple-negative breast cancer (TNBC), which is insensitive to the existing treatment regimens. However, the synergistic potency of traditional small-molecule combinations is limited in TNBC mainly due to mismatched molar ratios, inconsistent pharmacokinetics, and intratumoral accumulation of individual drugs. Here, we find that the autophagy inhibitor hydroxychloroquine (HCQ) and the topoisomerase I inhibitor 7-ethyl-10-hydroxycamptothecin (SN38) exhibit synergistic effects when the molar ratio reaches 5:1. We further develop a glutathione-responsive self-assembled combination nanoparticle (Combo NP) to integrate individual HCQ and SN38 polymeric prodrugs at the optimized ratio. In TNBC cells treated with Combo NP, HCQ-mediated autophagy blockage significantly enhances the DNA damage and apoptotic effect of SN38, manifesting synergistically cytotoxic effects of Combo NP. In vivo evaluations show that Combo NP maintains the molar ratio of HCQ to SN38 within the synergistic range in mouse blood circulation and intratumoral tissues. More importantly, Combo NP elicits superior therapeutic benefit in metastatic TNBC models, compared to free drug combination as well as single drug nanoparticles. Taken together, our engineered nanosystem highlights a nanoprodrug-based chemosensitizing approach for improving the therapeutic response to TNBC, addressing the major challenges of the current combination therapy.

Enhancing anaerobic digestion of pharmaceutical industries wastewater with the composite addition of zero valent iron (ZVI) and granular activated carbon (GAC).

Anaerobic digestion of pharmaceutical wastewater is challenged by its contained toxic compounds which limits the stability and efficiency of methane production and organic degradation. In this study, zero valent iron (ZVI) and granular activated carbon (GAC) were added with different strategies to improve anaerobic digestion of pharmaceutical wastewater. The results confirmed synergy effects of ZVI + GAC for both COD removal (increased by 13.4%) and methane production (increased by 11.0%). Furthermore, ZVI + GAC improved the removal of pharmaceutical intermediates, in particular, the residues (%) of dehydroepiandrosterone (DHEA) and 2,2'-methylenebis(6-tert-butyl-4-methylphenol) were only 30.48 ± 6.53 and 39.92 ± 4.50, and effectively reduced biotoxicity. The promoted results were attributed to the establishment of direct interspecies electron transfer (DIET). Microbial community analysis revealed that ZVI + GAC decreased species evenness and richness in bacterial whereas increased in archaeal. The relative abundance of acetotrophic methanogens decreased but hydrogenotrophic and methylotrophic methanogens increased, which broadening the pathway of methane production.

Selective Electrocatalytic Reduction of Nitrate to Ammonia with Nickel Phosphide.

Liquid ammonia is considered a sustainable liquid fuel and an easily transportable carrier of hydrogen energy; however, its synthesis processes are energy-consuming, high cost, and low yield rate. Herein, we report the electrocatalytic reduction of nitrate (NO3-) (ERN) to ammonia (NH3) with nickel phosphide (Ni2P) used as a noble metal-free cathode. Ni2P with (111) facet was grown in situ on nickel foam (NFP), which was regarded as a self-supporting cathode for ERN to synthesis NH3 with high yield rate (0.056 mmol h-1 mg-1) and superior faradaic efficiency of 99.23%. The derived atomic H (*H), verified by a quenching experiment and an electron spin resonance (ESR) technique, effectively enhanced the high selectivity for NH3 generation. DFT calculations indicated that *NO3 was deoxygenated to *NO2 and *NO, and *NO was subsequently hydrogenated with *H to generate NH3 with an energy releasing process (ΔG < 0). OLEMS also proved that NO was the merely gas intermediate. NFP exhibited the unique superhydrophilic surface, metallic properties, low impedance, and abundant surface sites, favorable for adsorption of NO3-, generation of *H, and then hydrogenation of NO3-. Hence, NFP cathode showed high selectivity for NH3 (89.1%) in ERN. NFP with long-term stability and low energy consumption provides a facile strategy for synthesis of NH3 and elimination of NO3- contamination.

Macrophage-Mediated Tumor Cell Phagocytosis: Opportunity for Nanomedicine Intervention.

Macrophages are one of the most abundant non-malignant cells in the tumor microenvironment, playing critical roles in mediating tumor immunity. As important innate immune cells, macrophages possess the potential to engulf tumor cells and present tumor-specific antigens for adaptive antitumor immunity induction, leading to growing interest in targeting macrophage phagocytosis for cancer immunotherapy. Nevertheless, live tumor cells have evolved to evade phagocytosis by macrophages via the extensive expression of anti-phagocytic molecules, such as CD47. In addition, macrophages also rapidly recognize and engulf apoptotic cells (efferocytosis) in the tumor microenvironment, which inhibits inflammatory responses and facilitates immune escape of tumor cells. Thus, intervention of macrophage phagocytosis by blocking anti-phagocytic signals on live tumor cells or inhibiting tumor efferocytosis presents a promising strategy for the development of cancer immunotherapies. Here, the regulation of macrophage-mediated tumor cell phagocytosis is first summarized, followed by an overview of strategies targeting macrophage phagocytosis for the development of antitumor therapies. Given the potential off-target effects associated with the administration of traditional therapeutics (for example, monoclonal antibodies, small molecule inhibitors), we highlight the opportunity for nanomedicine in macrophage phagocytosis intervention.

Relaxin gene delivery modulates macrophages to resolve cancer fibrosis and synergizes with immune checkpoint blockade therapy.

Cancer fibrosis serves as a major therapeutic barrier in desmoplastic tumors. Relaxin (RLN; a systemic hormone) is efficacious to decrease fibrosis, but the in vivo mechanism of action is not clear. Considering the localization of relaxin family peptide receptor type 1 (RXFP1), the receptor for RLN, on macrophages, we hypothesize that macrophages can be modulated by RLN to ameliorate cancer fibrosis. Using KPC mouse model of pancreatic ductal adenocarcinoma (PDAC), here, we report locally expressed RLN with targeted gene delivery induces increased F4/80+CD206+ macrophages originating from Ly6C+ monocytes, promoting fibrosis depletion and cytotoxic T cell infiltration. Moreover, RLN gene delivery synergizes with PD-L1 blockade for tumor inhibition by enhancing T cell-mediated tumor cell killing and macrophage phagocytosis. Collectively, our results reveal previously unidentified insights into the modulation of macrophages to regulate tumor-associated fibrosis, providing a feasible strategy to reverse the immunosuppressive environment and improve the therapeutic outcome of checkpoint immunotherapies.

Tumor-targeted gene therapy with lipid nanoparticles inhibits tumor-associated adipocytes and remodels the immunosuppressive tumor microenvironment in triple-negative breast cancer.

Adipocytes are the primary cellular components within the tumor microenvironment (TME) of triple-negative breast cancer (TNBC). Increasing evidence suggests that tumor-associated adipocytes (TAAs) can aggravate tumor progression, exacerbate the immunosuppressive TME and compromise therapeutic efficacy. In this study, the biological effect of TAAs within the breast cancer TME is first investigated, and the C-C Motif Chemokine Ligand 2 (CCL2) which is mainly secreted by TAAs in the extracellular environment is identified as the key mediator. CCL2 recruits immune cells such as monocytes and macrophages that further differentiated into immunosuppressive myeloid-derived suppressor cells (MDSCs) and M2 macrophages. To manipulate CCL2-mediated immune response, a protein trap that binds with CCL2 with high affinity and specificity is designed. The plasmid DNA encoding the CCL2 trap (pCCL2) is specifically delivered to the TME by using targeted lipid-protamine-DNA (LPD) nanoparticles to locally express the CCL2 trap and ameliorate the immunosuppressive TME. Significantly, compared with the commercially available CCL2 antibody, this strategy shows enhanced therapeutic efficacy and appreciable tumor growth inhibition. Furthermore, the pCCL2 trap treatment successfully suppresses TAAs, increases T cell infiltration and decreases the population of immunosuppressive M2 macrophages and MDSCs. Further studies show that the pCCL2 trap could facilitate PD-L1 blockade immunotherapy, demonstrating its translation potential.

Celastrol nanoemulsion induces immunogenicity and downregulates PD-L1 to boost abscopal effect in melanoma therapy.

Programmed cell death-ligand 1 (PD-L1)-based immune checkpoint blockade therapy using the anti-PD-L1 antibody is effective for a subset of patients with advanced metastatic melanoma but about half of the patients do not respond to the therapy because of the tumor immunosuppressive microenvironment. Immunogenic cell death (ICD) induced by cytotoxins such as doxorubicin (DOX) allows damaged dying tumor cells to release immunostimulatory danger signals to activate dendritic cells (DCs) and T-cells; however, DOX also makes tumor cells upregulate PD-L1 expression and thus deactivate T-cells via the PD-1/PD-L1 pathway. Herein, we show that celastrol (CEL) induced not only strong ICD but also downregulation of PD-L1 expression of tumor cells. Thus, CEL was able to simultaneously activate DCs and T-cells and interrupt the PD-1/PD-L1 pathway between T-cells and tumor cells. In a bilateral tumor model, intratumorally (i.t.) injected celastrol nanoemulsion retaining a high tumor CEL concentration activated the immune system efficiently, which inhibited both the treated tumor and the distant untreated tumor in the mice (i.e., abscopal effect). Thus, this work demonstrates a new and much cost-effective immunotherapy strategy - chemotherapy-induced immunotherapy against melanoma without the need for expensive immune-checkpoint inhibitors.

Nanoparticle-enhanced chemo-immunotherapy to trigger robust antitumor immunity.

Mounting evidence suggests that immunotherapies are a promising new class of anticancer therapies. However, the immunosuppressive tumor microenvironment (TME), poor immunogenicity, and off-target toxicity hinder the broader implementation of immunotherapies. Here, we describe a novel strategy combining chemotherapy and immunotherapy to modulate the TME by systemically and concurrently delivering the chemotherapeutic agent SN38 (7-ethyl-10-hydroxycamptothecin) and the STING agonist DMXAA (5,6-dimethylxanthenone-4-acetic acid) into tumors using triblock copolymer nanoparticles, named PS3D1@DMXAA, which enhances antigen cross-presentation and induces the conversion of the immunosuppressive TME to immunogenic TME through the newly found synergistic function between SN38 and STING activation. PS3D1@DMXAA thus shows potent therapeutic efficacy in three mice tumor models and elicits remarkable therapeutic benefit when combined with anti-PD-1 therapy. Our engineered nanosystem offers a rational design of an effective immunotherapy combination regimen to convert uninflamed "cold" tumors into "hot" tumors, addressing the major challenges immunotherapies faced.

Tumor neoantigen heterogeneity impacts bystander immune inhibition of pancreatic cancer growth.

The immunogenic clonal-fraction threshold in heterogeneous solid-tumor required to induce effective bystander-killing of non-immunogenic subclones is unknown. Pancreatic cancer poses crucial challenges for immune therapeutic interventions due to low mutational-burden and consequent lack of neoantigens. Here, we designed a model to incorporate artificial-neoantigens into genes-of -interest in cancer-cells and to test their potential to actuate bystander-killing. By precisely controlling a neoantigen's abundance in the tumor, we studied the impact of neoantigen frequency on immune-response and immune-escape. Our results showed single, strong, widely-expressed neoantigen could lead to robust antitumor response when over 80% of cancer cells express the neoantigen. Further, immunological assays demonstrated T-cell responses against non-target self-antigen on KRAS-oncoprotein, when we inoculated animals with a high frequency of tumor-cells expressing test-neoantigen. Using nanoparticle-based gene-therapy, we successfully altered tumor-microenvironment by perturbing interleukin-12 and interleukin-10 gene-expression. The subsequent microenvironment-remodeling reduced the neoantigen frequency threshold at which bioluminescent signal intensity for tumor-burden decreased 1.5-log-fold, marking robust tumor-growth inhibition, from 83% to 29%. Our results thus suggest bystander killing is inefficient in immunologically-cold tumors like pancreatic-cancer and requires high neoantigen abundance. However, bystander killing mediated antitumor response can be rescued by adjuvant-immune therapy.