Murine Colon Cancer Model Research Articles

AI-2 quorum sensing controlled delivery of cytolysin-A by tryptophan auxotrophic low-endotoxic Salmonella and its anticancer effects in CT26 mice with colon cancer

IntroductionThe limitations of conventional cancer therapies necessitate target-oriented, highly invasive, and safe treatment approaches. Hence, the intrinsic anti-tumor activity of Salmonella can offer better options to combat cancers. ObjectivesThis study aims to utilize attenuated Salmonella and deliver cytolytic protein cytolysin A (ClyA) under quorum sensing (QS) signaling for precise localized expression in tumors but not in healthy organs. MethodsThe therapeutic delivery strain was imposed with tryptophan auxotroph for selective colonization in tumors by trpA and trpE deletion, and lipid-A and O-antigen were altered by pagL and rfaL deletions using lambda red recombination method. The strain was transformed with the designed QS-controlled ClyA expression vector which was validated by western blot. The in vivo passaged therapeutic strain was used for treatment four times at a weekly interval, with a dose of 5 × 106 CFU/mouse for cancer therapy. ResultsThe attenuated strain induced minimal endotoxicity-related cytokines TNF-α, IL-1β, and IFN-γ and exhibited adequate colonization despite earlier exposure in mice. The QS-controlled ClyA expression was confirmed by western blot from bacterial cultures grown at different cell densities. The results demonstrated that the in vivo passaged strain preferentially colonized the tumor after vacating the spleen, liver, and lung, leaving no outward histological scars. The anti-cancer effect of the designed construct was evaluated in the murine CT26 colon cancer model. The expression of ClyA increased tumoricidal activity by 67 % compared to vector control. ConclusionHence, the anti-tumor effect of the engineered Salmonella strain was improved by ClyA expression via QS activation after achieving the threshold bacterial cell density. Further, immunohistochemical staining of the tumor and other organs corroborated the QS-controlled tumor-specific expression of ClyA. Overall, the results imply that the developed anti-cancer Salmonella has low endotoxicity and QS-controlled expression of ClyA as beneficial safety elements and supports recurrent Salmonella inoculation by O-antigen deficiency.

Antitumor effect of plant-produced anti-CTLA-4 monoclonal antibody in a murine model of colon cancer.

Cytotoxic T lymphocyte-associated protein 4 (CTLA-4) is an immune checkpoint regulator exclusively expressed on T cells that obstructs the cell's effector functions. Ipilimumab (Yervoy®), a CTLA-4 blocking antibody, emerged as a notable breakthrough in modern cancer treatment, showing upfront clinical benefits in multiple carcinomas. However, the exhilarating cost of checkpoint blockade therapy is discouraging and even utmost prominent in developing countries. Thereby, affordability of cancer care has become a point of emphasis in drug development pipelines. Plant expression system blossomed as a cutting-edge platform for rapid, facile to scale-up, and economical production of recombinant therapeutics. Here, we describe the production of an anti-CTLA-4 2C8 antibody in Nicotiana benthamiana. ELISA and bio-layer interferometry were used to analyze antigen binding and binding kinetics. Anticancer responses in vivo were evaluated using knocked-in mice implanted with syngeneic colon tumor. At 4 days post-infiltration, the antibody was transiently expressed in plants with yields of up to 39.65 ± 8.42 μg/g fresh weight. Plant-produced 2C8 binds to both human and murine CTLA-4, and the plant-produced IgG1 also binds to human FcγRIIIa (V158). In addition, the plant-produced 2C8 monoclonal antibody is as effective as Yervoy® in inhibiting tumor growth in vivo. In conclusion, our study underlines the applicability of plant platform to produce functional therapeutic antibodies with promising potential in cancer immunotherapy.

Inhibition of tumor intrinsic BANF1 activates antitumor immune responses via cGAS-STING and enhances the efficacy of PD-1 blockade

BackgroundBANF1 is well known as a natural opponent of cyclic GMP-AMP synthase (cGAS) activity on genomic self-DNA. However, the roles of BANF1 in tumor immunity remain unclear. Here, we investigate...

The Potency of Cowpea Mosaic Virus Particles for Cancer In Situ Vaccination Is Unaffected by the Specific Encapsidated Viral RNA.

Plant virus nanoparticles can be used as drug carriers, imaging reagents, vaccine carriers, and immune adjuvants in the formulation of intratumoral in situ cancer vaccines. One example is the cowpea mosaic virus (CPMV), a nonenveloped virus with a bipartite positive-strand RNA genome with each RNA packaged separately into identical protein capsids. Based on differences in their densities, the components carrying RNA-1 (6 kb) denoted as the bottom (B) component or carrying RNA-2 (3.5 kb) denoted as the middle (M) component can be separated from each other and from a top (T) component, which is devoid of any RNA. Previous preclinical mouse studies and canine cancer trials used mixed populations of CPMV (containing B, M, and T components), so it is unclear whether the particle types differ in their efficacies. It is known that the CPMV RNA genome contributes to immunostimulation by activation of TLR7. To determine whether the two RNA genomes that have different sizes and unrelated sequences cause different immune stimulation, we compared the therapeutic efficacies of B and M components and unfractionated CPMV in vitro and in mouse cancer models. We found that separated B and M particles behaved similarly to the mixed CPMV, activating innate immune cells to induce the secretion of pro-inflammatory cytokines such as IFNα, IFNγ, IL-6, and IL-12, while inhibiting immunosuppressive cytokines such as TGF-β and IL-10. In murine models of melanoma and colon cancer, the mixed and separated CPMV particles all significantly reduced tumor growth and prolonged survival with no significant difference. This shows that the specific RNA genomes similarly stimulate the immune system even though B particles have 40% more RNA than M particles; each CPMV particle type can be used as an effective adjuvant against cancer with the same efficacy as native mixed CPMV. From a translational point of view, the use of either B or M component vs the mixed CPMV formulation offers the advantage that separated B or M alone is noninfectious toward plants and thus provides agronomic safety.

Mouse CCL9 Chemokine Acts as Tumor Suppressor in a Murine Model of Colon Cancer.

Colorectal cancer is the third most frequently diagnosed cancer in the world. Despite extensive studies and apparent progress in modern strategies for disease control, the treatment options are still not sufficient and effective, mostly due to frequently encountered resistance to immunotherapy of colon cancer patients in common clinical practice. In our study, we aimed to uncover the CCL9 chemokine action employing the murine model of colon cancer to seek new, potential molecular targets that could be promising in the development of colon cancer therapy. Mouse CT26.CL25 colon cancer cell line was used for introducing lentivirus-mediated CCL9 overexpression. The blank control cell line contained an empty vector, while the cell line marked as CCL9+ carried the CCL9-overexpressing vector. Next, cancer cells with empty vector (control) or CCL9-overexpressing cells were injected subcutaneously, and the growing tumors were measured within 2 weeks. Surprisingly, CCL9 contributed to a decline in tumor growth in vivo but had no effect on CT26.CL25 cell proliferation or migration in vitro. Microarray analysis of the collected tumor tissues revealed upregulation of the immune system-related genes in the CCL9 group. Obtained results suggest that CCL9 reveals its anti-proliferative functions by interplay with host immune cells and mediators that were absent in the isolated, in vitro system. Under specific study conditions, we determined unknown features of the murine CCL9 that have so far bee reported to be predominantly pro-oncogenic.

Abstract 4235: Obesity drives inflammation and metabolic reprogramming in an orthotopic mouse model of early colon cancer

Abstract Obesity is strongly associated with colon cancer incidence and, in animal models, promotes colon cancer growth and progression. Mounting evidence indicates that circulating growth factors and chronic inflammation are procancer factors likely mediating this relationship. While obesity blunts antitumor immunity in many cancer types, how obesity-driven inflammation and reduced antitumor immunity may cooperate to promote colon cancer progression is not yet clear. Here we used a murine syngeneic orthotopic transplantation model of colon cancer to profile changes driven by diet-induced obesity (DIO) in the immune and cancer cell compartments of the tumor using digital spatial profiling and RNA sequencing. 7-9-week-old male and female C57BL/6 mice remained on either a 10 kcal% fat diet (control, n=34) or a 60 kcal% fat diet (DIO, n=27) for at least 19 weeks. 700 Apc-null;KrasG12D/+;Trp53-null;Smad4-null;tdTomato (i.e., AKPS) organoids were transplanted into the colonic wall of control and DIO mice via colonoscopy-guided injection. Tumors were excised after four weeks of growth, an early time point to assess changes between diet groups independent of tumor size differences or weight loss associated with tumor growth. RNA sequencing was performed on RNA extracted from tumoral CD45+ and EpCAM+ cell fractions isolated using Milltenyi MicroBeads. DIO mice weighed more than controls (males: controls 37.0 g ± 5.1 vs. DIO 47.4 g ± 4.3; females: controls 26.4 g ± 3.7 vs. DIO 45.9 g ± 9.7) and had more mesenteric fat surrounding the colon (males: controls 388 mg ± 217 vs. DIO 577 mg ± 185; females: controls 201 mg ± 143 vs 1176 mg ± 589). Leptin, resistin, and plasminogen activator inhibitor-1 were significantly elevated in the serum of DIO mice relative to control mice. Tumor weight did not differ between groups. Gene set enrichment analysis demonstrated that tumor cells from DIO mice were significantly enriched for pathways related to inflammation (INFLAMMATORY_RESPONSE, IL6_JAK_STAT3_SIGNALING), glucose and lipid metabolism (GLYCOLYSIS, FATTY_ACID_METABOLISM), and proliferation (E2F_TARGETS, G2M_CHECKPOINT). Within the CD45+ transcriptomic profiles, Tumor Immune Estimation Resource (TIMER2.0) indicated an increase in M0- and M1-like macrophages and B cells in tumors from DIO compared with control mice. Analysis of whole tumor sections from DIO mice showed significantly reduced CD3+ cells by immunofluorescence and more Ki67+ CD45+ immune cells by digital spatial profiling relative to tumors from control mice. Taken together, obesity drives an inflammatory profile in both the immune and epithelial cell populations of AKPS murine tumors, which is associated with increased markers of proliferation. Future studies will address whether inflammatory signals from the mesenteric fat adjacent to the colon tumors may contribute to obesity-driven tumor inflammation, and thereby to colon cancer progression. Citation Format: Elaine M. Glenny, Babak Mirminachi, Tori L. McFarlane, Jatin Roper, Stephen D. Hursting. Obesity drives inflammation and metabolic reprogramming in an orthotopic mouse model of early colon cancer. [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 4235.

Type 2 Dendritic Cells Orchestrate a Local Immune Circuit to Confer Antimetastatic Immunity.

The progression of transformed primary tumors to metastatic colonization is a lethal determinant of disease outcome. Although circulating adaptive and innate lymphocyte effector responses are required for effective antimetastatic immunity, whether tissue-resident immune circuits confer initial immunity at sites of metastatic dissemination remains ill defined. Here we examine the nature of local immune cell responses during early metastatic seeding in the lung using intracardiac injection to mimic monodispersed metastatic spread. Using syngeneic murine melanoma and colon cancer models, we demonstrate that lung-resident conventional type 2 dendritic cells (DC2) orchestrate a local immune circuit to confer host antimetastatic immunity. Tissue-specific ablation of lung DC2, and not peripheral DC populations, led to increased metastatic burden in the presence of an intact T cell and NK cell compartment. We demonstrate that DC nucleic acid sensing and transcription factors IRF3 and IRF7 signaling are required for early metastatic control and that DC2 serve as a robust source of proinflammatory cytokines in the lung. Critically, DC2 direct the local production of IFN-γ by lung-resident NK cells, which limits the initial metastatic burden. Collectively, our results highlight, to our knowledge, a novel DC2-NK cell axis that colocalizes around pioneering metastatic cells to orchestrate an early innate immune response program to limit initial metastatic burden in the lung.

CRISPR-mediated rapid arming of poxvirus vectors enables facile generation of the novel immunotherapeutic STINGPOX.

Poxvirus vectors represent versatile modalities for engineering novel vaccines and cancer immunotherapies. In addition to their oncolytic capacity and immunogenic influence, they can be readily engineered to express multiple large transgenes. However, the integration of multiple payloads into poxvirus genomes by traditional recombination-based approaches can be highly inefficient, time-consuming and cumbersome. Herein, we describe a simple, cost-effective approach to rapidly generate and purify a poxvirus vector with multiple transgenes. By utilizing a simple, modular CRISPR/Cas9 assisted-recombinant vaccinia virus engineering (CARVE) system, we demonstrate generation of a recombinant vaccinia virus expressing three distinct transgenes at three different loci in less than 1 week. We apply CARVE to rapidly generate a novel immunogenic vaccinia virus vector, which expresses a bacterial diadenylate cyclase. This novel vector, STINGPOX, produces cyclic di-AMP, a STING agonist, which drives IFN signaling critical to the anti-tumor immune response. We demonstrate that STINGPOX can drive IFN signaling in primary human cancer tissue explants. Using an immunocompetent murine colon cancer model, we demonstrate that intratumoral administration of STINGPOX in combination with checkpoint inhibitor, anti-PD1, promotes survival post-tumour challenge. These data demonstrate the utility of CRISPR/Cas9 in the rapid arming of poxvirus vectors with therapeutic payloads to create novel immunotherapies.

Effective combination of liposome-targeted chemotherapy and PD-L1 blockade of murine colon cancer.

Therapeutic cancer drug efficacy can be limited by insufficient tumor penetration, rapid clearance, systemic toxicity and (acquired) drug resistance. The poor therapeutic index due to inefficient drug penetration and rapid drug clearance and toxicity can be improved by using a liposomal platform. Drug resistance for instance against pemetrexed, can be reduced by combination with docetaxel. Here, we developed a specific liposomal formulation to simultaneously deliver docetaxel and pemetrexed to enhance efficacy and safety. Hydrophobic docetaxel and hydrophilic pemetrexed were co-encapsulated into pH-sensitive liposomes using a thin-film hydration method with high efficiency. The physicochemical properties, toxicity, and immunological effects of liposomes were examined in vitro. Biodistribution, anti-tumor efficacy, and systemic immune response were evaluated in vivo in combination with PD-L1 immune checkpoint therapy using two murine colon cancer models. In cellular experiments, the liposomes exhibited strong cytotoxicity and induced immunogenic cell death. In vivo, the treatment with the liposome-based drug combination inhibited tumor development and stimulated immune responses. Liposomal encapsulation significantly reduced systemic toxicity compared to the delivery of the free drug. Tumor control was strongly enhanced when combined with anti-PDL1 immunotherapy in immunocompetent mice carrying syngeneic MC38 or CT26 colon tumors. We showed that treatment with liposome-mediated chemotherapy of docetaxel and pemetrexed combined with anti-PD-L1 immunotherapy is a promising strategy for the treatment of colon cancers.

Immune Checkpoint Inhibitor-Mediated Cancer Theranostics with Radiolabeled Anti-Granzyme B Peptide.

Although immune checkpoint inhibitors (ICI) have revolutionized cancer management, patient response can be heterogeneous, and the development of ICI resistance is increasingly reported. Novel treatment strategies are necessary not only to expand the use of ICI to previously unresponsive tumor types but also to overcome resistance. Targeted radionuclide therapy may synergize well with ICIs since it can promote a pro-inflammatory tumor microenvironment. We investigated the use of a granzyme B targeted peptide (GZP) as a cancer theranostic agent, radiolabeled with 68Ga (68Ga-GZP) as a PET imaging agent and radiolabeled with 90Y (90Y-GZP) as a targeted radionuclide therapy agent for combinational therapy with ICI in murine models of colon cancer. Our results demonstrate that GZP increasingly accumulates in tumor tissue after ICI and that the combination of ICI with 90Y-GZP promotes a dose-dependent response, achieving curative response in some settings and increased overall survival.

Liposomal Formulations of a New Zinc(II) Complex Exhibiting High Therapeutic Potential in a Murine Colon Cancer Model.

Colorectal cancer is the second leading cause of cancer-related mortality. Many current therapies rely on chemotherapeutic agents with poor specificity for tumor cells. The clinical success of cisplatin has prompted the research and design of a huge number of metal-based complexes as potential chemotherapeutic agents. In this study, two zinc(II) complexes, [ZnL2] and [ZnL(AcO)], where AcO is acetate and L is an organic compound combining 8-hydroxyquinoline and a benzothiazole moiety, were developed and characterized. Analytical and spectroscopic studies, namely, NMR, FTIR, and UV-Vis allowed us to establish the complexes’ structures, demonstrating the ligand-binding versatility: tetradentate in [ZnL(AcO)] and bidentate in [ZnL2]. Complexes were screened in vitro using murine and human colon cancer cells cultured in 2D and 3D settings. In 2D cells, the IC50 values were <22 µM, while in 3D settings, much higher concentrations were required. [ZnL(AcO)] displayed more suitable antiproliferative properties than [ZnL2] and was chosen for further studies. Moreover, based on the weak selectivity of the zinc-based complex towards cancer cell lines in comparison to the non-tumorigenic cell line, its incorporation in long-blood-circulating liposomes was performed, aiming to improve its targetability. The resultant optimized liposomal nanoformulation presented an I.E. of 76% with a mean size under 130 nm and a neutral surface charge and released the metal complex in a pH-dependent manner. The antiproliferative properties of [ZnL(AcO)] were maintained after liposomal incorporation. Preliminary safety assays were carried out through hemolytic activity that never surpassed 2% for the free and liposomal forms of [ZnL(AcO)]. Finally, in a syngeneic murine colon cancer mouse model, while free [ZnL(AcO)] was not able to impair tumor progression, the respective liposomal nanoformulation was able to reduce the relative tumor volume in the same manner as the positive control 5-fluorouracil but, most importantly, using a dosage that was 3-fold lower. Overall, our results show that liposomes were able to solve the solubility issues of the new metal-based complex and target it to tumor sites.

Weight Loss and/or Sulindac Mitigate Obesity-associated Transcriptome, Microbiome, and Protumor Effects in a Murine Model of Colon Cancer.

Obesity is a colon cancer risk and/or progression factor, but the underlying mechanisms are incompletely understood. Herein we demonstrate that obesity enhances murine colon carcinogenesis and expression of numerous tumoral procancer and immunosuppressive pathways. Moreover, we establish that weight loss via LFD and/or the NSAID sulindac mitigate procancer effects of obesity.

Tumor-Microenvironment-Activated NIR-II Nanotheranostic Platform for Precise Diagnosis and Treatment of Colon Cancer.

Rational design of tumor-microenvironment (TME)-activated nanoformulation for precisely targeted cancer treatment has recently attracted an enormous attention. However, the all-in-one TME-activated theranostic nanosystems with a simple preparation and high biocompatibility are still rarely reported. Herein, catalase nanocrystals (CatCry) are first introduced as a tumor microenvironment activatable nanoplatform for selective theranostics of colon cancer. They are engaged as (i) a "nanoreactor" for silver nanoparticles (AgNP) synthesis, (ii) a nanovehicle for tumor delivery of anticancer drug doxorubicin (DOX), and (iii) an in situ O2 generator to relief tumor hypoxia. When CatCry-AgNP-DOX nanoformulation is within a tumor, the intratumoral H2S turns AgNP into Ag2S nanoparticles, inducing a photothermal effect and NIR-II emission under 808 nm laser irradiation and also triggering DOX release. Simultaneously, CatCry catalyzes intratumoral H2O2 into O2, relieving hypoxia and enhancing chemotherapy. In contrast, when delivered to healthy tissue without increased concentration of H2S, the developed nanoformulation remains in the "off" state and no theranostic action takes place. Studies with colon cancer cells in vitro and a murine colon cancer model in vivo demonstrated that CatCry-AgNP-DOX delivered a synergistic combination of PTT and enhanced chemotherapy, enabling complete eradication of tumor with minimal side effects. This work not only introduces nanoplatform for theranostics of H2S-rich tumors but also suggests a general strategy for protein-crystal-based nanomedicine.

PUMILIO proteins promote colorectal cancer growth via suppressing p21

PUMILIO (PUM) proteins belong to the highly conserved PUF family post-transcriptional regulators involved in diverse biological processes. However, their function in carcinogenesis remains under-explored. Here, we report that Pum1 and Pum2 display increased expression in human colorectal cancer (CRC). Intestine-specific knockout of Pum1 and Pum2 in mice significantly inhibits the progression of colitis-associated cancer in the AOM/DSS model. Knockout or knockdown of Pum1 and/or Pum2 in human CRC cells result in a significant decrease in the tumorigenicity and delayed G1/S transition. We identify p21/Cdkn1a as a direct target of PUM1. Abrogation of the PUM1 binding site in the p21 mRNA also results in decreased cancer cell growth and delayed G1/S transition. Furthermore, intravenous injection of nanoparticle-encapsulated anti-Pum1 and Pum2 siRNAs reduces colorectal tumor growth in murine orthotopic colon cancer models. These findings reveal the requirement of PUM proteins for CRC progression and their potential as therapeutic targets.

Fabrication and assessment of folic acid conjugated-chitosan modified PLGA nanoparticle for delivery of alpha terpineol in colon cancer

The objective of this study was to fabrication of α-terpineol-PLGA nanoparticles coated with folic acid-chitosan (αT-PCF-NPs) as well as evaluates their anticancer effects. αT-PCF-NPs were synthesized using the nanoprecipitation method and characterized by Dynamic light scattering (DLS), zeta potential (ZP), scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FTIR) analysis. Folic acid (FA) binding rate and entrapment efficiency of α-T were assessed by HPLC method. MTT assay was performed for cytotoxicity assessment. Quantitative polymerase chain reaction (qPCR) analysis, acridine orange and propodium iodide (AO/PI) staining and cell cycle analysis were done to assess the pro-apoptotic properties of αT-PCF-NPs. Molecular analysis for angiogenesis and antioxidant properties and murine colon cancer model for antitumor effects of αT-PCF-NPs were used. The % FA-binding and encapsulation efficiency of α-T in αT-PCF-NPs (particle size of 263.95 nm, polydispersity index (PDI) of 0.25, and surface charge of +38.20 mV) was reported to be 67% and 88.1% respectively. The higher inhibitory effect of αT-PCF-NPs on cancer cells compared to HFF cells was confirmed. The pro-apoptotic effect of αT-PCF-NPs was showed by increased SubG1 phase cells, AO/PI staining results and up and down regulation Bax and Bcl-2 as pro and anti-apoptotic genes in HT-29 cells. Antioxidant (SOD) and angiogenesis genes (VEGF and VEGF-R) were inhibited by αT-PCF-NPs exposure in HT-29 cells and also decreased the size of murine tumors was confirmed in exposure of αT-PCF-NPs. αT-PCF-NPs can be considered as a promising anticancer drug for colon cancer.

Imaging Kv1.3 Expressing Memory T Cells as a Marker of Immunotherapy Response.

Simple SummaryCancer immunotherapy has shown huge potential in the fight against cancer but few patients respond durably to treatment. Durable immunological responses are associated with the generation of memory T cells. A subset of memory T cells, effector memory T cells, have been shown to be associated with tumours responding to immune checkpoint inhibitor therapy in a range of cancer types. These effector memory T cells overexpress the potassium channel, Kv1.3, on the cell surface. In the current manuscript, we have developed a new Kv1.3 targeting peptide radiopharmaceutical, [18F]AlF-NOTA-KCNA3P, and characterised its in vivo biodistribution and ability to stratify response to immune checkpoint inhibitor therapy, correlating tumour uptake to changes in tumour associated immune cell populations. Overall, [18F]AlF-NOTA-KCNA3P successfully predicted response to immune checkpoint inhibitors in a murine model of colon cancer where tracer uptake in the tumour correlated well with changes in tumour associated memory T cell populations.Immune checkpoint inhibitors have shown great promise, emerging as a new pillar of treatment for cancer; however, only a relatively small proportion of recipients show a durable response to treatment. Strategies that reliably differentiate durably-responding tumours from non-responsive tumours are a critical unmet need. Persistent and durable immunological responses are associated with the generation of memory T cells. Effector memory T cells associated with tumour response to immune therapies are characterized by substantial upregulation of the potassium channel Kv1.3 after repeated antigen stimulation. We have developed a new Kv1.3 targeting radiopharmaceutical, [18F]AlF-NOTA-KCNA3P, and evaluated whether it can reliably differentiate tumours successfully responding to immune checkpoint inhibitor (ICI) therapy targeting PD-1 alone or combined with CLTA4. In a syngeneic colon cancer model, we compared tumour retention of [18F]AlF-NOTA-KCNA3P with changes in the tumour immune microenvironment determined by flow cytometry. Imaging with [18F]AlF-NOTA-KCNA3P reliably differentiated tumours responding to ICI therapy from non-responding tumours and was associated with substantial tumour infiltration of T cells, especially Kv1.3-expressing CD8+ effector memory T cells.

Granzyme B PET Imaging in Response to In Situ Vaccine Therapy Combined with αPD1 in a Murine Colon Cancer Model.

Immune checkpoint inhibitors (ICIs) block checkpoint receptors that tumours use for immune evasion, allowing immune cells to target and destroy cancer cells. Despite rapid advancements in immunotherapy, durable response rates to ICIs remains low. To address this, combination clinical trials are underway assessing whether adjuvants can enhance responsiveness by increasing tumour immunogenicity. CpG-oligodeoxynucleotides (CpG-ODN) are synthetic DNA fragments containing an unmethylated cysteine-guanosine motif that stimulate the innate and adaptive immune systems by engaging Toll-like receptor 9 (TLR9) present on the plasmacytoid dendritic cells (pDCs) and B cells. Here, we have assessed the ability of AlF-mNOTA-GZP, a peptide tracer targeting granzyme B, to serve as a PET imaging biomarker in response to CpG-ODN 1585 in situ vaccine therapy delivered intratumourally (IT) or intraperitoneally (IP) either as monotherapy or in combination with αPD1. [18F]AlF-mNOTA-GZP was able to differentiate treatment responders from non-responders based on tumour uptake. Furthermore, [18F]AlF-mNOTA-GZP showed positive associations with changes in tumour-associated lymphocytes expressing GZB, namely GZB+ CD8+ T cells, and decreases in suppressive F4/80+ cells. [18F]AlF-mNOTA-GZP tumour uptake was mediated by GZB expressing CD8+ cells and successfully stratifies therapy responders from non-responders, potentially acting as a non-invasive biomarker for ICIs and combination therapy evaluation in a clinical setting.

Cucurbitacin B controls M2 macrophage polarization to suppresses metastasis via targeting JAK-2/STAT3 signalling pathway in colorectal cancer

Ethnopharmacological relevanceCucurbitacin B (CuB), extracted from muskmelon pedicel, is a widely available triterpenoid molecule that exerts influence on various biological activities. Modern pharmacological studies have found that cucurbitacin B has many kinds of pharmacological anti-tumor and anti-metastasis functions. Aim of the studyTo explore the mechanism of anti-tumor and anti-metastasis effect of cucurbitacin B. Materials and methodsThe effect of cucurbitacin B on the growth of HCT116 and CT-26 was detected by CCK8; apoptosis was determined by flow cytometry and colony formation; the expression of apoptosis-related protein Bax, Bcl-2 and Cleaved-caspase-3 were examined by western Blot. To explore the underlying mechanism of cucurbitacin B against tumor, the Western blot, Immunofluorescence staining, Microscale Thermophoresis assays were used. Multiple molecular biology experiments were applied to validate the effect of polarization of cucurbitacin B-induced macrophages. The supernatant of Cucurbitacin B-induced macrophages and colon cells were co-cultured in vitro, and then transwell and wound healing assay were employed to the related phenotypes. C57BL/6 and BALB/c murine colon cancer model were also used to study the drug effects in vivo. ResultsCucurbitacin B distinctly induced the apoptosis of CRC cells. It was observed that cucurbitacin B not only inhibited the phosphorylation of JAK2 and STAT3, but also the translocation from the cytosol to the nucleus. Meanwhile, we observed that cucurbitacin B is bound to STAT3. Further experimentation demonstrated that cucurbitacin B reduced the polarization of M2 macrophage by down-regulating JAK2/STAT3 signaling pathway. Cucurbitacin B-induced M2-like macrophages were found to diminish the migration of CRC cells. In vitro study suggested that cucurbitacin inhibited the CRC cells proliferation via JAK2/STAT3 and suppressed the cell migration by suppressing M2-like macrophages polarization. Consistent with in vitro results, the cucurbitacin B therapy significantly inhibited tumor growth and metastasis in mice. Moreover, in vivo the treatment with cucurbitacin B enhanced anti-tumor immunity by regulating M2-like macrophages and promoted the expression of CD4 and CD8 in tumor microenvironment. ConclusionOur results proved that cucurbitacin B might be a potential candidate agent for adjuvant therapy in the process of CRC growth and metastasis.

TIPE2 Promotes Tumor Initiation But Inhibits Tumor Progression in Murine Colitis-Associated Colon Cancer.

Colorectal cancer (CRC) is the third leading cause of cancer in the United States, and inflammatory bowel disease patients have an increased risk of developing CRC due to chronic intestinal inflammation with it being the cause of death in 10% to 15% of inflammatory bowel disease patients. TIPE2 (TNF-alpha-induced protein 8-like 2) is a phospholipid transporter that is highly expressed in immune cells and is an important regulator of immune cell function. The azoxymethane/dextran sulfate sodium murine model of colitis-associated colon cancer (CAC) was employed in Tipe2 -/- and wild-type mice, along with colonoid studies, to determine the role of TIPE2 in CAC. Early on, loss of TIPE2 led to significantly less numbers of visible tumors, which was in line with its previously described role in myeloid-derived suppressor cells. However, as time went on, loss of TIPE2 promoted tumor progression, with larger tumors appearing in Tipe2 -/- mice. This was associated with increased interleukin-22/STAT3 phosphorylation signaling. Similar effects were also observed in primary colonoid cultures, together demonstrating that TIPE2 also directly regulated colonocytes in addition to immune cells. This work demonstrates that TIPE2 has dual effects in CAC. In the colonocytes, it works as a tumor suppressor. However, in the immune system, TIPE2 may promote tumorigenesis through suppressor cells or inhibit it through IL-22 secretion. Going forward, this work suggests that targeting TIPE2 for CRC therapy requires cell- and pathway-specific approaches and serves as a cautionary tale for immunotherapy approaches in general in terms of colon cancer, as intestinal inflammation can both promote and inhibit cancer.

Inulin coated Mn3O4 nanocuboids coupled with RNA interference reverse intestinal tumorigenesis in Apc knockout murine colon cancer models

This study reports the development and pre-clinical evaluation of biodrug using RNA interference and nanotechnology. The major challenges in achieving targeted gene silencing in vivo include the stability of RNA molecules, accumulation into pharmacological levels, and site-specific targeting of the tumor. We report the use of Inulin for coating the arginine stabilized manganese oxide nanocuboids (MNCs) for oral delivery of shRNA to the gut. Furthermore, bio-distribution analysis exhibited site-specific targeting in the intestines, improved pharmacokinetic properties, and faster elimination from the system without cytotoxicity. To evaluate the therapeutic possibility and effectiveness of this multimodal bio-drug, it was orally delivered to Apc knockout colon cancer mice models. Persistent and efficient delivery of bio-drug was demonstrated by the knockdown of target genes and increased median survival in the treated cohorts. This promising utility of RNAi-Nanotechnology approach advocates the use of bio-drug in an effort to replace chemo-drugs as the future of cancer therapeutics.