Colon Carcinoma Model Research Articles

Dual A2A/ A2B adenosine receptor antagonist M1069 counteracts immuno-suppressive mechanisms of adenosine and reduces tumor growth in vivo.

While A2A adenosine receptor (AR) was considered as a major contributor to adenosine-mediated immunosuppression, A2B, having the lowest affinity to adenosine, has also emerged as a potential contributor to tumor promotion. Therefore, in adenosine-rich tumor microenvironment (TME), where A2B could be complementary and/or compensatory to A2A, simultaneous targeting of A2A and A2B ARs can provide higher potential for cancer immunotherapy. We developed M1069 - a highly selective dual antagonist of the A2A and A2B AR. In assays with primary human and murine immune cells, M1069 rescued IL 2 production from T cells (A2A dependent) and inhibited VEGF production by myeloid cells (A2B dependent) in adenosine-high settings. M1069 also demonstrated superior suppression of secretion of pro tumorigenic cytokines CXCL1, CXCL5, and rescue of IL 12 secretion from adenosine differentiated dendritic cells compared to an A2A selective antagonist (A2Ai). In a one-way mixed lymphocyte reaction (MLR) assay, adenosine differentiated human and murine dendritic cells treated with M1069 demonstrated superior T cell stimulatory activity compared to dendritic cells differentiated in presence of A2Ai. In vivo, M1069 decreased tumor growth as a monotherapy and enhanced anti-tumor activity of bintrafusp alfa (BA) or cisplatin in syngeneic adenosinehi/CD73hi 4T1 breast tumor model, but not in the CD73 knockout (KO) 4T1 tumor model or in adenosinelow/CD73low MC38 murine colon carcinoma model. In summary, our dual A2A/A2B AR antagonist M1069 may counteract immune-suppressive mechanisms of high concentrations of adenosine in vitro and in vivo and enhance the anti-tumor activity of other agents, including BA and cisplatin.

Iron consumption strengthens anti-tumoral STING activation mediated by manganese-based nanoparticles

The cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway, as an important part in innate immunity, has recently emerged as a promising target for improving tumor therapy. Manganese ions (Mn2+) are an emerging agonist in the cGAS-STING pathway with multifaceted advantages, however manganese-based nanoparticles alone as the Mn2+ source have shown limited activity in eliciting anti-tumor immune responses compared to conventional organic STING agonists, and the underlying mechanism of the suboptimal efficiency remains unclear. Here, we demonstrate that intratumoral iron ions attenuate manganese-induced anti-tumor STING activation, and that the utilization of deferoxamine (DFO), an iron chelator that depletes intratumoral iron ions, effectively increases the intracellular accumulation of Mn2+ and thus promoted the STING activation efficiency of a hyaluronic acid modified manganese carbonate-silica hybrid nanoparticle (DS@Mn-H) in macrophages. The mechanism study suggests that the addition of DFO inhibited the expression of ferroportin (FPN), which serves as a Mn2+ exporter to reduce intracellular Mn2+ level. The synergistic effect of DS@Mn-H and DFO achieved excellent anti-tumor activities in a mouse colon carcinoma model. This work provides new insights on improving the Mn-based metallo-immunotherapy of cancer.

OMIP-105: A 30-color full-spectrum flow cytometry panel to characterize the immune cell landscape in spleen and tumor within a syngeneic MC-38 murine colon carcinoma model.

This panel was designed to characterize the immune cell landscape in the mouse tumor microenvironment as well as mouse lymphoid tissues (e.g., spleen). As an example, using the MC-38 mouse syngeneic tumor model, we demonstrated that we could measure the frequency and characterize the functional status of CD4 T cells, CD8 T cells, regulatory T cells, NK cells, B cells, macrophages, granulocytes, monocytes, and dendritic cells. This panel is especially useful for understanding the immune landscape in "cold" preclinical tumor models with very low immune cell infiltration and for investigating how therapeutic treatments may modulate the immune landscape.

The impact of phospholipids with high transition temperature to enhance redox-sensitive liposomal doxorubicin efficacy in colon carcinoma model

In this study, we have developed a redox-sensitive (RS) liposomal doxorubicin formulation by incorporating 10,10′-diselanediylbis decanoic acid (DDA) organoselenium compound as the RS moiety. Hence, several RS liposomal formulations were prepared by using DOPE, HSPC, DDA, mPEG2000-DSPE, and cholesterol. In situ drug loading using a pH gradient and citrate complex yielded high drug to lipid ratio and encapsulation efficiency (100%) for RS liposomes. Liposomal formulations were characterized in terms of size, surface charge and morphology, drug loading, release properties, cell uptake and cytotoxicity, as well as therapeutic efficacy in BALB/c mice bearing C26 tumor cells. The formulations showed an average particle size of 200 nm with narrow size distributions (PDI < 0.3), and negative surface charges varying from −6 mV to −18.6 mV. Our study confirms that the presence of the DDA compound in liposomes is highly sensitive to hydrogen peroxide at 0.1% w/v, resulting in a significant burst release of up to 40%. The in vivo therapeutic efficacy study in BALB/c mice bearing C26 colon carcinoma confirmed the promising function of RS liposomes in the tumor microenvironment which led to a prolonged median survival time (MST). The addition of hydrogenated soy phosphatidylcholine (HSPC) with a high transition temperature (Tm: 52–53.5°C) extended the MST of our 3-component formulation of F14 (DOPE/HSPC/DDA) to 60 days in comparison to Caelyx (PEGylated liposomal Dox), which is not RS-sensitive (39 days). Overall, HSPC liposomes bearing RS-sensitive moiety enhanced therapeutic efficacy against colon cancer in vitro and in vivo. This achievement unequivocally underscores the criticality of high-TM phospholipids, particularly HSPC, in significantly enhancing liposome stability within the bloodstream. In addition, RS liposomes enable the on-demand release of drugs, leveraging the redox environment of tumor cells, thereby augmenting the efficacy of the formulation.

CXCR4-Targeted Macrophage-Derived Biomimetic Hybrid Vesicle Nanoplatform for Enhanced Cancer Therapy through Codelivery of Manganese and Doxorubicin.

Immune-cell-derived membranes have garnered significant attention as innovative delivery modalities in cancer immunotherapy for their intrinsic immune-modulating functionalities and superior biocompatibilities. Integrating additional parental cell membranes or synthetic lipid vesicles into cellular vesicles can further potentiate their capacities to perform combinatorial pharmacological activities in activating antitumor immunity, thus providing insights into the potential of hybrid cellular vesicles as versatile delivery vehicles for cancer immunotherapy. Here, we have developed a macrophage-membrane-derived hybrid vesicle that has the dual functions of transporting immunotherapeutic drugs and shaping the polarization of tumor-associated macrophages for cancer immunotherapy. The platform combines M1 macrophage-membrane-derived vesicles with CXCR4-binding-peptide-conjugated liposomes loaded with manganese and doxorubicin. The hybrid nanovesicles exhibited remarkable macrophage-targeting capacity through the CXCR4-binding peptide, resulting in enhanced macrophage polarization to the antitumoral M1 phenotype characterized by proinflammatory cytokine release. The manganese/doxorubicin-loaded hybrid vesicles in the CXCR4-expressing tumor cells evoked potent cancer cytotoxicity, immunogenic cell death of tumor cells, and STING activation. Moreover, cotreatment with manganese and doxorubicin promoted dendritic cell maturation, enabling effective tumor growth inhibition. In murine models of CT26 colon carcinoma and 4T1 breast cancer, intravenous administration of the manganese/doxorubicin-loaded hybrid vesicles elicited robust tumor-suppressing activity at a low dosage without adverse systemic effects. Local administration of hybrid nanovesicles also induced an abscessive effect in a bilateral 4T1 tumor model. This study demonstrates a promising biomimetic manganese/doxorubicin-based hybrid nanovesicle platform for effective cancer immunotherapy tailored to the tumor microenvironment, which may offer an innovative approach to combinatorial immunotherapy.

Abstract 3298: Unlocking the potential of OPM-383: A novel LRRK2 inhibitor in cancer therapy

Abstract Background: Leucine-rich repeat kinase 2 (LRRK2) plays a pivotal role in regulating various cellular processes, such as cell proliferation, survival, and inflammation. LRRK2 exhibits dual functionality as a serine-threonine kinase and as a GTPase. It is involved in the modulation of multiple signaling pathways, including WNT, MAPK, NF-kB and mTOR. Germline mutations in LRRK2 are associated with an increased risk of cancer, particularly hormone-related and colorectal cancers. LRRK2 also promotes tumor cell growth and survival in papillary renal and thyroid carcinomas, DLBCL and cholangiocarcinoma cells. OPM has designed and developed a novel oral LRRK2 inhibitor, OPM383, using its proprietary Nanocyclix® technology. In this study, we have evaluated the pharmacokinetic (PK) properties, efficacy and tolerability of OPM383 in a colon carcinoma model and a panel of patient-derived organoids. Materials and Methods: In vitro potency of our Type I inhibitor, OPM383, against full-length wild-type LRRK2 and the G2019S pathogenic LRRK2 mutant was assessed in in a Lanthascreen cell assay. For PK studies, we analyzed OPM383 plasma levels using UPLVC/MS-MS. Evaluation of inhibition of LRRK2 phosphorylation (Ser935) was assessed by western blot. MC-38 cells were inoculated into C57BL/6 mice. When tumor masses reached 75 mm³, mice were randomized to receive OPM383 (50 mg/kg, orally, daily), anti-PD1 antibody (10 mg/kg, ip, twice weekly), or their combination. OPM383 was also evaluated in patient-derived organoids using the SEngine-Paris® platform. Results: OPM-383 inhibited wild-type LRRK2 and LRRK2[G2019S] with an IC50 of 42 nM and 33 nM, respectively. OPM383 is highly selective among a panel of &amp;gt;400 protein kinases (only 6.7% cross-reactivity at 100 nM). The compound is permeable, metabolically stable and possesses drug-like properties. OPM-383 exhibited a favorable pharmacokinetic profile in mice (AUC of 11858 h*ng/mL and T max of 3 hours at a dose of 30 mg/kg via oral administration). Preliminary e-ADME results indicated moderate inhibition of CYP3A4BFC but no significant inhibition of CYPs 1A2, 2D6, 3A4BQ, 2C19, and 2C9 by OPM-383. After 90 minutes of oral administration in CD1 mice, we observed the inhibition of LRRK2 phosphorylation (Ser935) in the brain, kidney, and PBMCs. In assessing its potential antitumor effects, OPM383 was tested on a panel of patient-derived organoids. Notably, OPM383 inhibited the proliferation of 7 out of 29 patient-derived organoids after 6 days of exposure at various concentrations (&amp;lt;10 µM). In mice bearing subcutaneous MC38 tumors, OPM383 significantly inhibited tumor growth compared to the vehicle-treated group. Conclusion: This study characterizes a potent first-in-class LRRK2 inhibitor in cancer models. The findings suggest that OPM383 could serve as an attractive lead scaffold for designing and synthesizing a new class of kinase inhibitors for potential applications in cancer therapy. Citation Format: Maria Eugenia Riveiro, Petra Blom, Kenji Shoji, Jan Hoflack. Unlocking the potential of OPM-383: A novel LRRK2 inhibitor in cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3298.

Abstract 4204: Subcutaneous vs. orthotopic tumor models: A comparative assessment

Abstract The failure of murine tumor models to adequately simulate the native biological milieu and tissue architecture of human malignancies, has fomented much discourse surrounding the discord that exists between experimental outcomes and clinical settings. Orthotopic (OT) models, which are generated by the engraftment of tumor cells or fragments into recipient organs of the same histotype from which they were derived, has the advantage of recapitulating the appropriate vasculature, as well as cellular and stromal components as the site of origin. In the context of immunotherapy, there is increasing evidence that subcutaneous (SC) tumors bear a vastly different immunological profile compared to OT implants, a phenomenon dictated by tissue-specific recruitment of various immune subsets, and subsequently, impacts overall response. Therefore, establishing cancer models that better represent the natural tumor environment is imperative for addressing some of the foibles inherent to preclinical mouse models. To demonstrate the significance of the anatomical site of the tumor, we present a comparative assessment of the MC38 murine colon carcinoma model, in the context of SC and OT settings. In the SC model of MC38, administration of either 5mg/kg anti-mPD1, anti-mPDL1, or anti-mCTLA-4, failed to elicit any notable anti-tumor activity, as evidenced by 0% incidences of complete regressions (CR) and tumor free survivors (TFS) for all three therapeutic agents, and incidences of 11%, 5% and -22% increase in time to progression (ITP), respectively. However, mice bearing OT tumors, exhibited meaningful response to 5mg/kg anti-mPD1 and anti-mPDL1, both evoking incidences of 25% CR, 25% TFS and 108% ITP. Though the anti-mCTLA-4 treated cohort did not produce any CRs or TFS, there was, nonetheless, a 50% incidence of ITP. Taken together, the data clearly illustrates the distinct disparities in response between SC and OT tumors, with respect to immunomodulatory agents. Characterization of immune phenotype within both settings, and interrogation of additional mouse models are ongoing. The exigencies and profound limitations associated with bench to bedside translation have long encumbered the drug development process. Despite staggering financial investment in oncology research, effective treatments for a myriad of cancers remain elusive, and only 3.4% of cancer drug candidates are conferred with clinical approval. Employing modalities that adequately reproduce the molecular and structural frameworks of human tumors, is crucial to better elucidating the natural trajectory of cancer progression, thus facilitating more accurate evaluation of drug activity and therapeutic response in preclinical settings. OT models therefore represent a more disease-relevant preclinical approach compared to their subcutaneous counterparts, as they more reliably preserve the intrinsic infrastructure and complexities of patient tumors in situ. Citation Format: Kerry-Ann Bright, Sheri Barnes, Derrik Germain, Lauren Kucharczyk. Subcutaneous vs. orthotopic tumor models: A comparative assessment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4204.

Abstract 3255: Targeting CD4+FOXP3+ Tregs to enhance anti-tumor immunity

Abstract We strive to improve the outcomes of cancer immunotherapy by using a new generation of chemically modified self-delivering antisense oligonucleotides (ASO), termed FANA ASO, to target the key T-regulatory (Treg) cell transcription factor, FOXP3. We tested five murine FANA in 2 murine tumor models (TC1, MC38) and studied 19 human FANA FOXP3 in vitro with human healthy donor PBMC, followed by use of clinical samples from lung cancer patients and patients with mesothelioma, as well as testing in humanized mice (hu-PBMC-NSG). In murine studies, we treated mice on day 7 after tumor inoculation with Scramble control or murine FANA FOXP3, 50 mg/kg for 14 days, i.p. In the TC1 lung tumor model, we found an average 38% decrease of tumor volumes, p = 0.0007, and 22% of tumors were completely resorbed. In the MC38 colon carcinoma model, we found a 49% decrease of tumor volumes, p = 0.0182, and 13.6% of tumors were completely resorbed. In both tumor models, we observed an ~50% decrease of Foxp3 mRNA by qPCR, and decreased numbers of intratumoral Tregs by flow cytometry. This was accompanied by significant decreases (p&amp;lt;0.05) of the mRNA and protein intratumoral levels of multiple exhaustion markers: LAG-3, CTLA-4, PD-1, Tim-3, Tigit, CD244, CD160 and VISTA, and increased expression of perforin and granzyme-b by intratumoral T cells. In addition, there were no changes in FOXP3 mRNA expression or in the numbers of Tregs in draining lymph nodes and in spleens of tumor bearing mice, suggesting that intratumoral Treg have enhanced sensitivity to FANA FOXP3 in vivo. We identified the best 5 of 19 human FANA FOXP3 and showed these had no toxic effects on cell viability or division, and downregulated FOXP3 expression over 50% in PBMC from healthy donors (p&amp;lt;0.05). Human Treg, pre-incubated with FANA FOXP3 for 3 hours, retained only 60.3% of their suppressive function in vitro. FANA FOXP3 treatment of lung cancer tumor samples and mesothelioma pleural effusion samples resulted in a 56.4% decrease in FOXP3 mRNA expression and a 61.5% decrease in Treg numbers (all p&amp;lt;0.05). Moreover, tumor FOXP3+ Treg had 36.7% less FOXP3 protein per cell, and downregulated CD39 expression compared to Scramble control. Targeting of cancer samples with human FANA FOXP3 in vitro was associated with increased division of CD4+ and CD8+ cells, and with downregulation of multiple exhaustion molecules, including CTLA-4, Tim-3, PD-1, LAG-3 and TIGIT. Humanized mice, treated with two human FANA FOXP3 compounds (50 mg/kg, 7days), demonstrated an absence of apparent toxic effects and human FOXP3 mRNA was downregulated by 47.4% in the blood, by 74.2% in lymph nodes and by 51.8% in the spleens (all p&amp;lt;0.05). Hence, targeting of intratumoral Tregs using FANA FOXP3 results in enhanced immune responses to solid tumors without inducing autoimmunity, and studies are underway in combination with checkpoint inhibitors and/or CAR-T cell therapy. Citation Format: Tatiana Akimova, Liqing Wang, Zhanna Bartosh, Evgeniy Eruslanov, Steven Albelda, Sunil Singhal, Veenu Aishwarya, Wayne W. Hancock. Targeting CD4+FOXP3+ Tregs to enhance anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3255.

Abstract 3984: Full-length TCR and BCR repertoire analysis, integrated with mRNA and surface proteins characterization, in a tumor mouse model

Abstract Full-length TCR and BCR analysis with single-cell multiomic assays provides an understanding of immune responses, offering insights into the complete receptor structure and functionality that is crucial for accurately assessing the diversity and specificity of immune repertoires. Studying mouse immune cell heterogeneity can be achieved by simultaneous mRNA and surface protein profiling. However, when coupled with full-length TCR and BCR analysis, this integrated approach provides a more comprehensive understanding of immune responses and cellular function in healthy and diseased conditions. In this study, we used the MC38 colon carcinoma model to study T and B cell heterogeneity and clonotype diversity. We stained single-cell suspensions prepared from bone marrow, thymus, spleen and tumor tissues with 20 AbSeq (CITE-Seq) antibody oligos and sample-multiplexing tags. We then loaded 50,000 isolated T and B cells into two lanes of an 8-lane microwell cartridge for single-cell capture by a single-cell analysis system. To aid in the simultaneous analysis of clonality and gene expression, we carried out library preparation using a full-length TCR/BCR multiomic assay and a mouse immune response panel. The combination of mRNA and protein expression confirmed the elevated expression of exhaustion and activation markers in tumor-infiltrating lymphocytes (TILs). We identified high-frequency clonotypes within TILs and tracked their frequency across other tissues. Tumor-burdened mice splenic T cells showed shared TCR combinations with TILs, which were absent in healthy tissue. The results show the ability to successfully profile T and B cell receptors using a full-length TCR/BCR multiomic assay in a mouse tumor model and the use of transcriptome, protein and TCR/BCR clonal information to investigate immune cell characteristics across multiple samples. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo and BD Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2023 BD. All rights reserved. NPM-2536 (v1.0) 1023 Citation Format: Ali Rezvan, Xiaoshan Shi, Moen Sen, Stephanie Widmann, Aaron Tyznik. Full-length TCR and BCR repertoire analysis, integrated with mRNA and surface proteins characterization, in a tumor mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3984.

Abstract 4041: Translational assessment of triple combination with Tislelizumab (anti-PD-1), LBL-007 (anti-LAG-3) and &amp;lt; for Surzebiclimab (anti-TIM-3) highlights its strong anti-tumor activity and &amp;lt; for clinical potential in solid tumors such as HNSCC

Abstract Background: Therapeutics targeting PD-(L)1 have demonstrated impressive clinical activity in several type of cancers. However, only a small proportion of patients develop long-term response and resistance frequently occurs. LAG-3 and TIM-3 are inhibitory immune checkpoints frequently upregulated and co-expressed with PD-1 on tumor-infiltrating T cells, contributing to T cell dysfunction. Thus, we hypothesized that simultaneously targeting PD-1/LAG-3/TIM-3 would further restore T cell response and the triple combination of Tislelizumab (Tisle, anti-PD-1), LBL-007 (anti-LAG-3) and Surzebiclimab (Surze, anti-TIM-3) would provide greater clinical benefit.Methods: To evaluate the rationale and the anti-tumor activity of triple combination, LAG-3 and TIM-3 expression were analyzed in in vitro T cells, syngeneic mouse models and cancer patients with anti-PD-(L)1 therapy. The effects of dual blockade of PD-1/LAG-3, PD-1/TIM-3 and the triple blockade of PD-1/LAG-3/TIM-3 on T cell function were evaluated in in vitro activated PBMCs and the anti-tumor efficacy were evaluated in syngeneic mouse models. Relevant gene expression and immune signatures were ranked across 31 solid tumor types in TCGA.Results: Both LAG-3 and TIM-3 expression on T cells were upregulated by anti-PD-1 treatment in syngeneic mouse tumors, similar trend was observed in cancer patients with anti-PD-(L)1 therapy. Dual combination of Tisle/LBL-007 or Tisle/Surze enhanced IFNγ production in in vitro activated human T cells. The dual blockade of PD-1/LAG-3 or PD-1/TIM-3 significantly enhanced tumor growth inhibition compared with anti-PD-1 monotherapy in syngeneic mouse models. The triple combination of Tisle/LBL-007/Surze further enhanced IFNγ production in in vitro activated T cells. In a mouse MC38 colon carcinoma model, the triple blockade of PD-1/LAG-3/TIM-3 demonstrated enhanced anti-tumor activity compared with either dual combination, evidenced by trend of increased tumor growth inhibition and higher tumor-free incidence rate. Finally, the responsiveness of PD-1/LAG-3/TIM-3 triple combination was predicted using 5 signatures, including early effector T cell signature, inflamed signature, LAG-3, TIM-3 and PD-L1 expression. The in silico analyses indicated strong potential in clinical utility such as squamous cell carcinoma of the head and neck (HNSCC).Conclusions: The concurrent blockade of PD-1/LAG-3/TIM-3 represents a promising strategy to enhance T cell function and anti-tumor activity. The results demonstrated the therapeutic potential of the triple combination. A Ph2 study evaluating Tisle in combination with LBL-007 and/or Surze in first-line treatment of recurrent or metastatic HNSCC (NCT05909904) is recruiting. Citation Format: Hengrui Zhu, Jinhui Zhang, Han Yan, Xiao Ding, Juan Yang, Yu Jiang, Minjuan Deng, Haoxuan Song, Fangfang Ping, Fuyun Sun, Xiaoyu Li, Lijie Zhang, Bin Jiang, Weiwei Song, Zhirong Shen, Wei Jin, Jiyuan Zhang, Yun Zhang. Translational assessment of triple combination with Tislelizumab (anti-PD-1), LBL-007 (anti-LAG-3) and &amp;lt; for Surzebiclimab (anti-TIM-3) highlights its strong anti-tumor activity and &amp;lt; for clinical potential in solid tumors such as HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4041.

A Cancer Nanovaccine for Co-Delivery of Peptide Neoantigens and Optimized Combinations of STING and TLR4 Agonists.

Immune checkpoint blockade (ICB) has revolutionized cancer treatment and led to complete and durable responses, but only for a minority of patients. Resistance to ICB can largely be attributed to insufficient number and/or function of antitumor CD8+ T cells in the tumor microenvironment. Neoantigen targeted cancer vaccines can activate and expand the antitumor T cell repertoire, but historically, clinical responses have been poor because immunity against peptide antigens is typically weak, resulting in insufficient activation of CD8+ cytotoxic T cells. Herein, we describe a nanoparticle vaccine platform that can overcome these barriers in several ways. First, the vaccine can be reproducibly formulated using a scalable confined impingement jet mixing method to coload a variety of physicochemically diverse peptide antigens and multiple vaccine adjuvants into pH-responsive, vesicular nanoparticles that are monodisperse and less than 100 nm in diameter. Using this approach, we encapsulated synergistically acting adjuvants, cGAMP and monophosphoryl lipid A (MPLA), into the nanocarrier to induce a robust and tailored innate immune response that increased peptide antigen immunogenicity. We found that incorporating both adjuvants into the nanovaccine synergistically enhanced expression of dendritic cell costimulatory markers, pro-inflammatory cytokine secretion, and peptide antigen cross-presentation. Additionally, the nanoparticle delivery increased lymph node accumulation and uptake of peptide antigen by dendritic cells in the draining lymph node. Consequently, nanoparticle codelivery of peptide antigen, cGAMP, and MPLA enhanced the antigen-specific CD8+ T cell response and delayed tumor growth in several mouse models. Finally, the nanoparticle platform improved the efficacy of ICB immunotherapy in a murine colon carcinoma model. This work establishes a versatile nanoparticle vaccine platform for codelivery of peptide neoantigens and synergistic adjuvants to enhance responses to cancer vaccines.

Unlocking PD-1 antibody resistance: The MUC1 DNA vaccine augments CD8+ T cell infiltration and attenuates tumour suppression.

In light of increasing resistance to PD1 antibody therapy among certain patient populations, there is a critical need for in-depth research. Our study assesses the synergistic effects of a MUC1 DNA vaccine and PD1 antibody for surmounting PD1 resistance, employing a murine CT26/MUC1 colon carcinoma model for this purpose. When given as a standalone treatment, PD1 antibodies showed no impact on tumour growth. Additionally, there was no change observed in the intra-tumoural T-cell ratios or in the functionality of T-cells. In contrast, the sole administration of a MUC1 DNA vaccine markedly boosted the cytotoxicity of CD8+ T cells by elevating IFN-γ and granzyme B production. Our compelling evidence highlights that combination therapy more effectively inhibited tumour growth and prolonged survival compared to either monotherapy, thus mitigating the limitations intrinsic to single-agent therapies. This enhanced efficacy was driven by a significant alteration in the tumour microenvironment, skewing it towards pro-immunogenic conditions. This assertion is backed by a raised CD8+/CD4+ T-cell ratio and a decrease in immunosuppressive MDSC and Treg cell populations. On the mechanistic front, the synergistic therapy amplified expression levels of CXCL13 in tumours, subsequently facilitating T-cell ingress into the tumour setting. In summary, our findings advocate for integrated therapy as a potent mechanism for surmounting PD1 antibody resistance, capitalizing on improved T-cell functionality and infiltration. This investigation affords critical perspectives on enhancing anti-tumour immunity through the application of innovative therapeutic strategies.

Enhanced antitumour response of gold nanostar-mediated photothermal therapy in combination with immunotherapy in a mouse model of colon carcinoma.

This study investigated the potential of combining PTT with dendritic cell (DC)-based immunotherapy and anti-PD-L1 immune checkpoint blockade (ICB) therapy against colorectal cancer and elucidated the underlying mechanisms. The CT26 tumour-bearing mice were divided into seven treatment groups: control, atezolizumab (A), dendritic cells (DC), pAuNSs-mediated PTT (PTT), PTT combined with atezolizumab (PTT + A), PTT combined with dendritic cells (PTT + DC), and PTT combined with dendritic cells and atezolizumab (PTT + DC + A). Therapeutic efficacy was monitored. PTT upregulated most immune cell membrane receptor genes, including PD-L1, and downregulated genes associated with antigen presentation and T cell activation. Although the PTT + A and PTT + DC treatments showed partial tumour growth retardation, the combination of PTT with DCs and atezolizumab (PTT + DC + A) exhibited the most significant antitumour effect, with a complete remission rate of 50% and prolonged survival. On day 14, tumour samples from non-responsive mice revealed insufficient recruitment of T cells as the reason for uncured tumours. Notably, mice cured with PTT + DC and PTT + DC + A treatments showed no detectable lung nodules. This study demonstrated that the combination of PTT with DC-based immunotherapy and atezolizumab effectively overcomes the non-sensitive nature of CT26 tumours. These findings highlight the potential of this combination approach for colorectal cancer treatment.

Macrophage membrane-coated self-assembled curcumin nanoparticle missile for the treatment of colorectal cancer

Curcumin is a natural compound with various anti-cancer properties, but its poor solubility and low bioavailability limit its clinical application. In this study, we developed a novel nanoparticle system based on curcumin self-assembly, polydopamine modification and macrophage membrane coating (Cur-PDA@CM) to overcome these challenges. The Cur-PDA@CM nanoparticles exhibited high drug loading, enhanced stability, excellent photothermal effects and powerful anti-tumor efficacy. Moreover, the macrophage membrane endows the nanoparticles with active targeting ability to tumor tissues. We evaluated the anti-tumor efficacy of Cur-PDA@CM in vitro and in vivo using a murine colon carcinoma model. The results showed that Cur-PDA@CM could effectively inhibit tumor growth and induce apoptosis by releasing curcumin, generating reactive oxygen species and polarizing tumor-associated macrophages towards the M1 phenotype. The combination of Cur-PDA@CM with near-infrared irradiation further enhanced the therapeutic outcomes by facilitating drug release and photothermal therapy. The Cur-PDA@CM system represents a promising platform for delivering natural products with poor solubility and harnessing their full anti-cancer potential.

Carbon Ion and Photon Radiation Therapy Show Enhanced Antitumoral Therapeutic Efficacy With Neoantigen RNA-LPX Vaccines in Preclinical Colon Carcinoma Models

Personalized liposome-formulated mRNA vaccines (RNA-LPX) are a new powerful tool in cancer immunotherapy. In preclinical tumor models, RNA-LPX are known to achieve potent results when combined together with conventional X-ray radiotherapy (XRT). Densely ionizing radiation used in carbon-ion radiotherapy (CIRT) may induce distinct effects in combination with immunotherapy, compared to sparsely ionizing X-rays. Within this study, we investigate the potential of CIRT and isoeffective doses of XRT to mediate tumor growth inhibition and survival in murine colon adenocarcinoma models in conjunction with neoantigen (neoAg)-specific RNA-LPX vaccines, encoding both MHC class I and II-restricted tumor-specific neoantigens. We characterize tumor immune infiltrates and antigen-specific T cell responses by flow cytometry and IFNγ ELISpot respectively. RNA-LPX vaccines significantly potentiate radiotherapy-mediated tumor growth inhibition. CIRT and XRT alone marginally prime neoAg-specific T cell responses in the tumor, but not in the blood or spleen of the mice. The infiltration and cytotoxicity of neoAg-specific T cells is strongly driven by RNA-LPX vaccines and accompanied by reduced expression of the inhibitory markers PD-1 and Tim-3 on these cells. NeoAg RNA-LPX vaccines shows similar overall therapeutic efficacy in combination with both CIRT and XRT, even if the physical radiation dose is lower for carbon ions than X-rays. We hence conclude that the combination of CIRT and neoAg RNA-LPX is a promising strategy for treatment of radioresistant tumors.

Tumor Regression upon Intratumoral and Subcutaneous Dosing of the STING Agonist ALG-031048 in Mouse Efficacy Models.

Stimulator of interferon genes (STING) agonists have shown potent anti-tumor efficacy in various mouse tumor models and have the potential to overcome resistance to immune checkpoint inhibitors (ICI) by linking the innate and acquired immune systems. First-generation STING agonists are administered intratumorally; however, a systemic delivery route would greatly expand the clinical use of STING agonists. Biochemical and cell-based experiments, as well as syngeneic mouse efficacy models, were used to demonstrate the anti-tumoral activity of ALG-031048, a novel STING agonist. In vitro, ALG-031048 is highly stable in plasma and liver microsomes and is resistant to degradation via phosphodiesterases. The high stability in biological matrices translated to good cellular potency in a HEK 293 STING R232 reporter assay, efficient activation and maturation of primary human dendritic cells and monocytes, as well as long-lasting, antigen-specific anti-tumor activity in up to 90% of animals in the CT26 mouse colon carcinoma model. Significant reductions in tumor growth were observed in two syngeneic mouse tumor models following subcutaneous administration. Combinations of ALG-031048 and ICIs further enhanced the in vivo anti-tumor activity. This initial demonstration of anti-tumor activity after systemic administration of ALG-031048 warrants further investigation, while the combination of systemically administered ALG-031048 with ICIs offers an attractive approach to overcome key limitations of ICIs in the clinic.

Immunotherapy with STING and TLR9 agonists promotes synergistic therapeutic efficacy with suppressed cancer-associated fibroblasts in colon carcinoma.

The innate immune sensing of nucleic acids using effective immunoadjuvants is critical for increasing protective immune responses against cancer. Stimulators of interferon genes (STING) and toll-like receptor 9 (TLR9) agonists are considered promising candidates in several preclinical tumor models with the potential to be used in clinical settings. However, the effects of such treatment on tumor stroma are currently unknown. In this study, we investigated the immunotherapeutic effects of ADU-S100 as a STING agonist and CpG ODN1826 as a TLR9 agonist in a preclinical model of colon carcinoma. Tumor-bearing mice were treated intratumorally on days 10 and 16 post-tumor inoculation with ADU-S100 and CpG ODN1826. Cytokine profiles in the tumor and spleen, tumor cell apoptosis, the infiltration of immune cells, and cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) were evaluated to identify the immunological mechanisms after treatment. The powerful antitumor activity of single and combination treatments, the upregulation of the expression of pro-inflammatory cytokines in the tumor and spleen, and the recruitment and infiltration of the TME by immune cells revealed the synergism of immunoadjuvants in the eradication of the colon carcinoma model. Remarkably, the significant downregulation of CAFs in the TME indicated that suppression of tumorigenesis occurred after immunoadjuvant therapy. The results illustrate the potential of targeting the STING and TLR9 pathways as powerful immunoadjuvants in the treatment of preclinical colon carcinoma and the possibility of harnessing these pathways in future therapeutic approaches.

IL-2/αIL-2 Complexes Massively Expand Systemic Tumor-Specific T Cells and Enhance Abscopal Responses to Radiation and αPD-1

Early clinical trials have provided evidence for RT-induced systemic effects in conjunction with αPD1 or IL-2 in metastatic patients, but strong abscopal responses are clinically rare. We investigated the effect of a triple combination of CD122-directed IL-2/αIL-2 complexes (IL-2c), hRT, and αPD1 on tumor-specific CD8+ T cell differentiation and the abscopal effect vs. the respective dual treatments. Mice bearing bilateral tumors were treated with two fractions of 8 Gy (C51 colon carcinoma model) or 12 Gy (B16 melanoma model); αPD1 was given weekly; IL-2c was given for five consecutive days. Dependence of the therapeutic effect on CD8+ T cells and T cells expressing the chemokine receptor CXCR3 was assessed using depleting or blocking antibodies. Differentiation stages of tumor-specific CD8+ T cells in various compartments were determined flow cytometrically using MHC-I tetramers and appropriate antibodies. Anti-tumoral effects of blood-derived and tumor-derived T cells were assessed in adoptive T cell transfer experiments. The abscopal effect was significantly stronger in triple-treated mice compared to mice treated with RT/αPD1 (C51 model: p < 0.01; B16 model: p < 0.05), RT/IL-2c (C51 model: p < 0.01; B16 model: p < 0.001) or αPD1/IL-2c (C51 model: p < 0.0001, B16 model: p < 0.01). Triple therapy improved survival and resulted in complete cures of 3/12 mice in the C51 model and 2/12 mice in the B16 model. These anti-tumor effects were associated with dramatic expansion of tumor-specific CD8+ T cells. Undifferentiated stem-like (TCF1+TIM3-PD1+) and effector-like (CD101-TIM3+TCF1-PD1+) but not terminally differentiated (CD101+TIM3+TCF1-PD1+) exhausted cells particularly strongly increased. Moreover, IL-2c induced CXCR3 mainly on non-terminally differentiated CD8+ T cells. Both CD8+ (C51 model: p < 0.0001; B16 model: p < 0.01) and CXCR3+ (C51 model: p < 0.0001) T cells were crucial for the RT-induced abscopal effect upon RT/αPD1/IL-2c treatment. Finally, we found that peripheral blood from triple-treated mice is an effective source of T cells for adoptive T cell transfer. RT/αPD1/IL-2c triple treatment resulted in superior local and systemic expansion of tumor-specific CD8+ T cells with stem- and effector-like phenotypes. Also, IL-2c strongly increased CXCR3+CD8+ T cells that were associated with pronounced abscopal responses in models with an established metastasis resistant to αPD1/IL-2c and only transiently responding to RT/αPD1 or RT/IL-2c. Therefore, such triple combinations appear promising for clinical evaluation in metastatic patients.

Molecularly Imprinted Nanomedicine for Anti-angiogenic Cancer Therapy via Blocking Vascular Endothelial Growth Factor Signaling.

The VEGF-VEGFR2 (VEGF = vascular endothelial growth factor) signaling has been a promising target in cancer therapy. However, because conventional anti-angiogenic therapeutics suffer from drawbacks, particularly severe side effects, novel anti-angiogenic strategies are much needed. Herein, we report the rational engineering of VEGF-targeted molecularly imprinted polymer nanoparticles (nanoMIP) for anti-angiogenic cancer therapy. The anti-VEGF nanomedicine was prepared via a state-of-the-art molecular imprinting approach using the N-terminal epitope of VEGF as the template. The nanoMIP could target the two major pro-angiogenic isoforms (VEGF165 and VEGF121) with high affinity and thereby effectively block the VEGF-VEGFR2 signaling, yielding a potent anti-angiogenic effect of "killing two birds with one stone". In vivo experiments demonstrated that the anti-VEGF nanoMIP effectively suppressed tumor growth via anti-angiogenesis in a xenograft model of human colon carcinoma without apparent side effects. Thus, this study not only proposes an unprecedented anti-angiogenic strategy for cancer therapy but also provides a new paradigm for the rational development of MIPs-based "drug-free" nanomedicines.

The CX3CL1-CX3CR1 chemokine axis can contribute to tumor immune evasion and blockade with a novel CX3CR1 monoclonal antibody enhances response to anti-PD-1 immunotherapy.

CX3CL1 secreted in the tumor microenvironment serves as a chemoattractant playing a critical role in metastasis of CX3CR1 expressing cancer cells. CX3CR1 can be expressed in both cancer and immune-inhibitory myeloid cells to facilitate their migration. We generated a novel monoclonal antibody against mouse CX3CR1 that binds to CX3CR1 and blocks the CX3CL1-CX3CR1 interaction. We next explored the immune evasion strategies implemented by the CX3CL1-CX3CR1 axis and find that it initiates a resistance program in cancer cells that results in 1) facilitation of tumor cell migration, 2) secretion of soluble mediators to generate a pro-metastatic niche, 3) secretion of soluble mediators to attract myeloid populations, and 4) generation of tumor-inflammasome. The CX3CR1 monoclonal antibody reduces migration of tumor cells and decreases secretion of immune suppressive soluble mediators by tumor cells. In combination with anti-PD-1 immunotherapy, this CX3CR1 monoclonal antibody enhances survival in an immunocompetent mouse colon carcinoma model through a decrease in tumor-promoting myeloid populations. Thus, this axis is involved in the mechanisms of resistance to anti-PD-1 immunotherapy and the combination therapy can overcome a portion of the resistance mechanisms to anti-PD-1.