Colorectal Carcinoma Model Research Articles

Cationic Metal-Organic Layer Delivers siRNAs to Overcome Radioresistance and Potentiate Cancer Radiotherapy.

Radiotherapy plays an important role in modern oncology, but its treatment efficacy is limited by the radioresistance of tumor cells. As a member of the inhibitor of apoptosis protein family, survivin plays a key role in developing radioresistance by mediating apoptosis evasion, promoting epithelial-mesenchymal transition, and modulating cell cycle dynamics. Efficient downregulation of survivin expression presents a promising strategy to enhance the antitumor effects of radiotherapy. Herein, we report the design of a hafnium-porphyrin-based cationic metal-organic layer (CMOL) with quaternary ammonium capping groups to deliver small interfering RNAs (siRNAs) for enhanced radiotherapy. The CMOL@siRNA nanoplatform not only increased energy deposition from X-rays and reactive oxygen species generation via a unique radiotherapy-radiodynamic therapy process, but also effectively delivered siRNAs to downregulate survivin expression and ameliorate radioresistance of cancer cells. Consequently, CMOL@siRNA in combination with low-dose X-ray irradiation demonstrated remarkable antitumor efficacy with 96.9% and 91.4% tumor growth inhibition in murine colorectal carcinoma and triple-negative breast cancer models, respectively.

Non-pathogenic E. coli displaying decoy-resistant IL18 mutein boosts anti-tumor and CAR NK cell responses.

The tumor microenvironment can inhibit the efficacy of cancer therapies through mechanisms such as poor trafficking and exhaustion of immune cells. Here, to address this challenge, we exploited the safety, tumor tropism and ease of genetic manipulation of non-pathogenic Escherichia coli (E. coli) to deliver key immune-activating cytokines to tumors via surface display on the outer membrane of E. coli K-12 DH5α. Non-pathogenic E. coli expressing murine decoy-resistant IL18 mutein (DR18) induced robust CD8+ T and natural killer (NK) cell-dependent immune responses and suppressed tumor progression in immune-competent colorectal carcinoma and melanoma mouse models. E. coli K-12 DH5α engineered to display human DR18 potently activated mesothelin-targeting chimeric antigen receptor (CAR) NK cells and enhance their trafficking into tumors, which extended survival in an NK cell treatment-resistant mesothelioma xenograft model by enhancing TNF signaling and upregulating NK activation markers. Our live bacteria-based immunotherapeutic system safely and effectively induces potent anti-tumor responses in treatment-resistant solid tumors, motivating further evaluation of this approach in the clinic.

Bi-specific antibody engagers for cancer immunotherapy.

Bispecific antibody engagers are fusion proteins composed of a nanobody that recognizes immunoglobulin kappa light chains (VHH kappa ) and a nanobody that recognizes either CTLA-4 or PD-L1. These fusions show strong antitumor activity in mice through recruitment of polyclonal immunoglobulins independently of specificity or isotype. In the MC38 mouse model of colorectal carcinoma, the anti-CTLA-4 VHH-VHH kappa conjugate eradicates tumors and reduces the number of intratumoral regulatory T cells. The anti-PD-L1 VHH-VHH kappa conjugate is less effective in the MC38 model, whilst still outperforming an antibody of similar specificity. The potency of the anti-PD-L1 VHH-VHH kappa conjugate was strongly enhanced by installation of the cytotoxic drug maytansine or a STING agonist. The ability of such fusions to engage the Fc-mediated functions of all immunoglobulin isotypes is an appealing strategy to further improve on the efficacy of immune checkpoint blockade, commonly delivered as a monoclonal immunoglobulin of a single defined isotype.

A laponite-based immunologically active gel delivery system for long-acting tumor vaccine

Traditional bolus vaccines typically require multiple doses, which complicates the vaccination process and may cause missed shots, leading to sub-optimal immunity and reduced vaccine effectiveness. Herein, a gel-based long-acting vaccine system with self-adjuvant properties based on laponite was constructed to simplify vaccination procedures and improve vaccine effectiveness. Firstly, the gel system could recruit multiple types of immune cells to form immune niches. Secondly, it could achieve sustained delivery of antigens to lymph nodes by active transport and passive drainage. Then, the gel system triggered the formation of a large number of germinal centers, which elicited enhanced and durable humoral immune responses, as well as strong cellular immune responses. As a result, it eventually showed good prophylactic and therapeutic effects in a variety of tumor models including melanoma, colorectal cancer and peritoneal metastasis models. By further combining the immunoadjuvant CpG ODN and cytokine IL-12, the effect of the gel-vaccine could be further enhanced. In a murine peritoneal metastasis model of colorectal carcinoma, a single administration of the gel-vaccine resulted in complete tumor eradication in 8/9 mice. In summary, this study developed an immunologically active gel-vaccine system. And as a robust and versatile vaccine platform, by loading different antigens and adjuvants, this gel-vaccine system is expected to realize its better therapeutic potential.

Lectin-Based Immunophenotyping and Whole Proteomic Profiling of CT-26 Colon Carcinoma Murine Model.

A murine colorectal carcinoma (CRC) model was established. CT26 colon carcinoma cells were injected into BALB/c mice's spleen to study the primary tumor and the mechanisms of cell spread of colon cancer to the liver. The CRC was verified by the immunohistochemistry of Pan Cytokeratin and Vimentin expression. Immunophenotyping of leukocytes isolated from CRC-bearing BALB/c mice or healthy controls, such as CD19+ B cells, CD11+ myeloid cells, and CD3+ T cells, was carried out using fluorochrome-labeled lectins. The binding of six lectins to white blood cells, such as galectin-1 (Gal1), siglec-1 (Sig1), Sambucus nigra lectin (SNA), Aleuria aurantia lectin (AAL), Phytolacca americana lectin (PWM), and galectin-3 (Gal3), was assayed. Flow cytometric analysis of the splenocytes revealed the increased binding of SNA, and AAL to CD3 + T cells and CD11b myeloid cells; and increased siglec-1 and AAL binding to CD19 B cells of the tumor-bearing mice. The whole proteomic analysis of the established CRC-bearing liver and spleen versus healthy tissues identified differentially expressed proteins, characteristic of the primary or secondary CRC tissues. KEGG Gene Ontology bioinformatic analysis delineated the established murine CRC characteristic protein interaction networks, biological pathways, and cellular processes involved in CRC. Galectin-1 and S100A4 were identified as upregulated proteins in the primary and secondary CT26 tumor tissues, and these were previously reported to contribute to the poor prognosis of CRC patients. Modelling the development of liver colonization of CRC by the injection of CT26 cells into the spleen may facilitate the understanding of carcinogenesis in human CRC and contribute to the development of novel therapeutic strategies.

Abstract 3323: Novel Pan-RAS inhibitor ADT-007: A unique mechanism of antitumor selectivity in pancreatic and colorectal carcinoma models

Abstract Multiple types of RAS mutations activate RAS isoforms, HRAS, NRAS, or KRAS, that drive oncogenic signaling leading to uncontrolled cell proliferation and tumor development. RAS mutations occur de novo in approximately one-third of all human cancers, and are especially prevalent in pancreatic, colorectal, and lung tumors. Where RAS mutations are less frequent in other cancers, such as breast cancer, RAS can be pathologically activated by growth factor receptors that engage numerous downstream effectors. The currently FDA-approved KRAS-targeted drugs are designed to covalently inactivate only G12C KRAS mutants, however, this mutation is only prevalent in lung cancer. The most common mutations in pancreatic ductal adenocarcinoma (PDAC) are KRAS-G12D, KRAS-G12V, and KRAS-G12R. Our novel class of pan-RAS inhibitors, exemplified by ADT-007, addresses the urgent need for pan-RAS inhibitors with antitumor activity in cancers harboring these common mutations. These pan-RAS inhibitors have unique pharmacological properties that allow for selective killing of cancer cells harboring RAS mutations. ADT-007 inhibited growth of an array of carcinoma tumor cell lines in vitro with single digit nM IC50 values regardless of RAS mutant allele, and inhibited RAS activation and MAPK/AKT signaling. Because it is a pan-RAS inhibitor, ADT-007 may not be susceptible to commonly reported mechanisms of resistance to covalent KRAS inhibitors, such as activation of wild type (WT) alleles or secondary RAS mutations. Differentiated normal epithelial or hepatic cells, and tumor cells with WT RAS were insensitive to ADT-007 due to enzymatic detoxification of ADT-007 via glucuronidation, representing a novel mechanism of tumor selectivity. An orally bioavailable prodrug of ADT-007 (ADT-1004) is well tolerated in vivo, and produced sustained concentrations of ADT-007 well above IC50 values in plasma and even higher levels in tumors. Daily oral administration of ADT-1004 significantly inhibited RAS signaling and tumor growth in orthotopic and PDX mouse models of pancreatic cancer. Citation Format: Junwei Wang, Adam B. Keeton, Sindhu Ramesh, Peyton Johnson, Yulia Y. Maxuitenko, Jeremy B. Foote, Chung-Hui Huang, Kristy L. Berry, Khalda Fadlalla, Bandi D. Reddy, Ganji P. Nagaraju, Elmar Nurmemmedov, Ivan Babic, Vadim Gaponenko, Xi Chen, Bassel F. El-Rayes, Donald J. Buchsbaum, Gary A. Piazza. Novel Pan-RAS inhibitor ADT-007: A unique mechanism of antitumor selectivity in pancreatic and colorectal carcinoma models [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 3323.

Abstract 2605: STX-1, a potent first-in-class ADC directed against DPP4 (CD26) as a biomarker of senescent tumor cells

Abstract Dipeptidyl Peptidase 4 (DPP4/CD26) is a membrane glycoprotein involved in particular chemotactic and inflammatory responses, via its N-terminal protein cleavage enzymatic activities. DPP4 expression is found in various cell types, in certain subsets of lymphocytes and frequently overexpressed in primary tumors and metastases where its role remains still poorly understood. Recently, DPP4 was identified as a novel biomarker of senescent cells. In cancers, the harmful effect of senescent tumor cells is well recognized and mediated by the local production of pro-inflammatory cytokines, soluble immunosuppressive factors and metalloproteinases, called ‘SASP’ phenotype which could be induced by many chemotherapy and radiation regimens. This deleterious microenvironment promotes tumor growth, invasion and neoangiogenesis which can lead to treatment resistance, relapses and poor prognosis in many cancers, especially at advanced stages. STX-1 is a leading first-in-class ADC platform based on a proprietary anti-DPP4 mAb generated after a screen between normal and senescence-induced cells identifying DPP4 as a candidate for cancer targeting. Other biomarkers of interest were also identified as being selectively expressed for further preclinical evaluation. This IgG1/k mAb has potent in vitro activities on DPP4+ cells: i) in a bleomycin-induced senescent fibroblasts model; ii) on DPP4-expressing and chemotherapy- or radiotherapy-induced senescent human tumor cell lines with overexpression of DPP4; iii) binding of the mAb to its target results in its internalization into the endolysosomal compartment of cells. The first formulations of STX-1 are undergoing preclinical evaluation in vitro and in vivo and consist of a classical maleimide-based conjugation, an enzymatically cleavable cathepsin-dipeptide linker comprising a self-immolative spacer and either inhibitor of tubulin (MMAE/DAR4) or topoisomerase I inhibitor (exatecan/DAR8) payloads. Plasma stability and affinity, dose-response potency and permeability bystander activity on human tumor cell lines were evaluated in vitro. Preliminary efficacy is demonstrated in vivo on tumor xenograft models with various DPP4 expressions (constitutive or chemo/radio-induced to senescence) based on a 21-28 days weekly infusion schedule (3 or 5 µg/kg). The indications selected for further therapeutic development are prostate, liver and colorectal carcinoma models. Data on antitumor efficacy, tolerability and pharmacokinetics will be presented. Further studies will include optimization of the best STX-1 formulation, NHP studies and GMP scale-up with planned IND submission expected in Q3-2024. Exploiting the targeting of senescent cells in cancer based on selected biomarkers such as DPP4 constitutes a new therapeutic avenue to counteract tumor dormancy, recurrences and resistance to conventional chemo/radiotherapies. Citation Format: Eric Angevin, Virginie Lelarge, Remi Capelle, Justine Choeur, Thierry Mathieu, Benjamin Le Calvé. STX-1, a potent first-in-class ADC directed against DPP4 (CD26) as a biomarker of senescent tumor cells [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 2605.

Abstract 3338: Development of a CK2α inhibitor for treatment of wnt-driven colorectal cancer

Abstract A novel, potent and highly selective orally bioavailable small molecule inhibitor of Casein Kinase 2α (CK2α) has been developed and validated in pre-clinical models of wnt-driven colorectal carcinoma; and is poised to enter clinical development.CK2α is considered as a positive regulator of wnt signaling and tumorigenesis. The wnt pathway is known to be sensitive to and amplified by CK2α activity at multiple nodes and can be inhibited by loss of CK2α function. The human genetics of colorectal cancer (CRC) have been well characterized and approximately 80% tumors are identified as being wnt pathway mutation driven. CRC tumors can be further stratified into consensus molecular subtypes (CMS), with iCMS2 representing 37% of CRC patients, being characterized by ongoing wnt pathway upregulation. Development of CK2α inhibitors as a targeted therapy for CRC is therefore mechanistically well aligned to the wnt signaling drive and may serve as a promising therapeutic strategy for this cancer. A novel small molecule binding site on CK2α has been exploited to design and develop an inhibitor with Ki of 95 pM and exquisite selectivity over the kinome. This inhibitor has been comprehensively characterized using in vitro, ex vivo and in vivo pharmacology studies. Cellular mechanism of action studies in CRC cell lines have quantified the potency of target engagement, inhibition of known CK2α phospho-sites, inhibition of tumorigenic wnt pathway activity, and induction of apoptosis. This CK2α inhibitor was also profiled in a high content screen of CRC patient-derived organoids established from various driver mutation backgrounds. Use of these ex vivo models has validated the potent cytotoxic activity of CK2α inhibition in tumor samples with wnt-driven characteristics and highlights the potential therapeutic efficacy of CK2α inhibition in this setting. In vivo CRC xenograft studies have confirmed anti-tumor efficacy and developed the understanding of pharmacokinetic pharmacodynamic relationship between free plasma concentration of drug and inhibition of CK2α-specific biomarker in the tumor. The preclinical data presented establishes the strong rationale for clinical development of a novel CK2α inhibitor for the treatment of locally advanced or metastatic solid tumors, with an emphasis on wnt-driven CRC. Citation Format: Darren Cawkill. Development of a CK2α inhibitor for treatment of wnt-driven colorectal cancer [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 3338.

Abstract 3185: Glatiramer acetate enhances tumor retention and mitigates systemic toxicity of toll-like receptor 9 agonists as intratumoral immunotherapy

Abstract Pathogen-associated molecular patterns (PAMP) are conserved molecular structures produced by microorganisms that are recognized as foreign by receptors of the innate immune system. Unmethylated cytosine-guanine oligodeoxynucleotides (CpG ODNs) are a storied drug class from the PAMP family that ligates Toll-like receptor 9 (TLR9). CpG ODNs have shown promising antitumor effects in preclinical studies by inducing potent pro-inflammatory immune responses. However, there are still practical challenges that have hindered clinical success, including inefficient in vivo delivery and severe immune-related toxicities caused by systemic exposure to CpG. We previously identified that glatiramer acetate (GA), an FDA-approved, lysine-rich polypeptide, could be applied as a cationic drug delivery vector for CpG delivery proximal to the injection site while mitigating systemic release of pro-inflammatory cytokines. To dive into the mechanism and evaluate pharmacological efficacy in more pathological models that simulate colorectal carcinoma, we validated the biodistribution of GA-CpG complexes (or R4B) and explored their safety and antitumor immunity in multiple mouse colorectal carcinoma models. In the CT26 single tumor model, intratumoral R4B treatment displayed comparable antitumoral efficacy as CpG treatment. Genome-wide transcriptome analysis showed that R4B treatment elicited much more prominent immune system response pathways in both lesions and spleens. The biodistribution study proved that R4B localized and gradually released CpG around the injection site while ‘naked’ CpG diffused away from the injection site quickly. In the CT26 dual tumor-bearing model, intratumoral administration of R4B generated systemic immune efficacy that led to an abscopal effect on distant untreated cancers, comparable to treatment with CpG alone. Notably, R4B treatment accomplished these effects with greatly mitigated systemic pro-inflammatory cytokines compared to CpG alone. These data help further elucidate the noncanonical role of glatiramer acetate to serve as a nucleic acid delivery scaffold that potentiates the safety of CpG adjuvant for cancer immunotherapy. Citation Format: Huan Gong, Daniel J. Griffin, Chad E. Groer, Xiaoqing Wu, Moustafa M. Abdelaziz, Sa Wu, Liang Xu, Laird M. Forrest, Cory J. Berkland. Glatiramer acetate enhances tumor retention and mitigates systemic toxicity of toll-like receptor 9 agonists as intratumoral immunotherapy [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 3185.

Canonical DDR activation by EMT inducing agent 5-Fluorouracil is modulated by a cannabinoid based combinatorial approach via inducing autophagy and suppression of vimentin expression

Anastasis cascade including induction of Epithelial to Mesenchymal Transition (EMT), DNA repair, and stimulation of pro-survival mediators collectively exaggerate therapy resistance in cancer prognosis. The extensive implications of DNA-damaging agents are clinically proven futile for the rapid development of disease recurrence during treatment regime. Herein we report a glycosidic derivative of Δ9-tetrahydrocannabinol (THC-9-OG) abrogates sub-toxic doses of 5-Fluorouracil (5FU) induced EMT in colon cancer cells nullifying DNA repairing mechanism. Our in vitro and in vivo data strongly proclaims that THC-9-OG could not only abrogate 5FU mediated background EMT activation through stalling matrix degradation as well as murine 4T1 lung metastasis but also vigorously diminished Rad-51 repairing mediator along with stimulation of γ-H2AX foci formation. The combinatorial treatment (5FU + THC-9-OG) in Apc knockout colorectal carcinoma model conferred remission of the crypt progenitor phenotype which was prominently identified in 5FU treatment. Mechanistically, we demonstrated that 5FU plus THC-9-OG significantly attenuated major EMT inducer Vimentin via extensive ROS generation along with autophagy induction via LC3B I-II conversion and p62 degradation in a p-ATM dependent manner. Additionally, Cannabinoid receptor CB1 was responsible for abrogation of Vimentin since we found increase in the expression of γH2AX and decrease in vimentin expression in CB1 agonist (ACEA) plus 5FU treated cells. Nutshell, our results unveil a new direction of Cannabinoid based combinatorial approach to control background EMT along with robust enhancing of DNA damage potential of sub-toxic concentration of 5FU resulting immense inhibition of distant metastasis coupled with triggering cell death in vitro and in vivo.

Antitumoral effect of local injection of TLR-9 agonist emulsified in Lipiodol with systemic anti-PD-1 in a murine model of colorectal carcinoma.

Local treatments of cancer, including transarterial chemoembolization, could enhance responses to systemic immune checkpoint inhibitors such as anti-PD-1 antibodies. Lipiodol, a radiopaque oil, is widely used for transarterial chemoembolization as a tumor-targeting drug carrier and could be used in emulsion with immunomodulators. This study aimed at evaluating the antitumoral effect of intra-tumoral injection of Lipiodol-immunomodulator emulsions combined with systemic anti-PD-1 therapy in a murine model of colorectal carcinoma. Mice (male BALB/c) with anti-PD-1-resistant subcutaneous CT26 tumors were injected with immunomodulators, emulsified or not with Lipiodol (N=10-12/group). The TLR-9 agonist CpG displayed antitumor effects, while Poly I:C and QS21 did not. The Lipiodol-CpG emulsion appeared to be stable and maintained CpG within tumors for a longer time. Repeated intra-tumoral injections, combined with anti-PD-1, induced responses towards the tumor as well as to a distant metastatic-like nodule. This treatment was associated with an increase in proliferative CD8+ T cells and of IFN-γ expression, a decrease in proliferative regulatory T cells but also, surprisingly, an increase in myeloid derived suppressor cells. Local administration of CpG emulsified with Lipiodol led to an effective antitumoral effect when combined to systemic anti-PD-1 therapy. Lipiodol, apart from its radiopaque properties, is an efficient drug-delivery system. The formulated oil-in-water emulsion allows efficient loading and control release of CpG, which induces favorable immune modifications in this murine tumor model.

Orthotopic MC-38 Allograft as a Robust Preclinical Model of Colorectal Carcinoma.

Colorectal cancer (CRC) is a significant global health concern, requiring effective preclinical models for studying its development and testing therapies. Mouse models have been used, including spontaneous tumors, carcinogen exposure, and tumor cell implantation as xenografts or at orthotopic sites. Here, we describe an orthotopic preclinical model of CRC, which provides a valuable tool for studying tumor growth and the tumor microenvironment, offering a more accurate representation of human CRC compared to xenograft models.

MCT4 blockade increases the efficacy of immune checkpoint blockade

Background & AimsIntratumoral lactate accumulation and acidosis impair T-cell function and antitumor immunity. Interestingly, expression of the lactate transporter monocarboxylate transporter (MCT) 4, but not MCT1, turned out to be...

Oncometabolite D-2-hydroxyglutarate—dependent metabolic reprogramming induces skeletal muscle atrophy during cancer cachexia

Cancer cachexia is characterized by weight loss and skeletal muscle wasting. Based on the up-regulation of catabolism and down-regulation of anabolism, here we showed genetic mutation-mediated metabolic reprogramming in the progression of cancer cachexia by screening for metabolites and investigating their direct effect on muscle atrophy. Treatment with 93 μM D-2-hydroxyglutarate (D2HG) resulted in reduced myotube width and increased expression of E3 ubiquitin ligases. Isocitrate Dehydrogenase 1 (IDH1) mutant patients had higher D2HG than non-mutant patients. In the in vivo murine cancer cachexia model, mutant IDH1 in CT26 cancer cells accelerated cachexia progression and worsened overall survival. Transcriptomics and metabolomics revealed a distinct D2HG-induced metabolic imbalance. Treatment with the IDH1 inhibitor ivosidenib delayed the progression of cancer cachexia in murine GL261 glioma model and CT26 colorectal carcinoma models. These data demonstrate the contribution of IDH1 mutation mediated D2HG accumulation to the progression of cancer cachexia and highlight the individualized treatment of IDH1 mutation associated cancer cachexia.

Deuterium Magnetic Resonance Imaging Using Deuterated Water-Induced 2H-Tissue Labeling Allows Monitoring Cancer Treatment at Clinical Field Strength.

An accurate and noninvasive assessment of tumor response following treatment other than traditional anatomical imaging techniques is essential. Deuterium magnetic resonance spectroscopic (MRS) imaging has been demonstrated as an alternative for cancer metabolic imaging by high-field MRI using deuterium-labeled molecules. The study aim was to use 2H tissue labeling and deuterium MRI at clinical field strength for tumor visualization and assessment of three anticancer therapies in pancreatic cancer model mice. MIA PaCa-2 pancreatic carcinoma and C26 colorectal carcinoma models of BALB/c-nu mice was prepared, and repeated deuterium MRI was performed during the first 10 days of free drinking of 30% D2O to track 2H distribution in tissues. 2H accumulation in the tumor after irradiation, bevacizumab administration, or gemcitabine administration was also measured in MIA PaCa-2-bearing mice. Confirmatory proton MRI, ex vivo metabolic hyperpolarization 13C-MRS, and histopathology were performed. The mouse's whole-body distribution of 2H was visible 1 day after drinking, and the signal intensity increased daily. Although the tumor size did not change 1 and 3 days after irradiation, the amount of 2H decreased significantly. The 2H image intensity of the tumor also significantly decreased after the administration of bevacizumab or gemcitabine. Metabolic hyperpolarization 13C-MRS, proton MRI, and 2H-NMR spectroscopy confirmed the efficacy of the anticancer treatments. Deuterium MRI at 1.5T proved feasible to track 2H distribution throughout mouse tissues during D2O administration and revealed a higher 2H accumulation in the tumor xenografts. This research demonstrated a promising successful method for preliminary assessment of radiotherapy and chemotherapy of cancer.

Sirtuin 2 up-regulation suppresses the anti-tumour activity of exhausted natural killer cells in mesenteric lymph nodes in murine colorectal carcinoma.

Natural killer (NK) cells inhibit colorectal carcinoma (CRC) initiation and progression through their tumoricidal activity. However, cumulative evidence suggests that NK cells become functionally exhausted in patients with CRC. To deepen the understanding of the mechanisms underlying CRC-associated NK cell exhaustion, we explored the expression and effect of Sirtuin 2 (Sirt2) in mesenteric lymph node (mLN) NK cells in a murine colitis-associated CRC model. Sirt2 was remarkably up-regulated in mLN NK cells after CRC induction. Particularly, Sirt2 was increased in mLN NK cells expressing high T cell immunoglobulin and mucin domain-3 (TIM3), high lymphocyte activation protein-3 (LAG3), high programmed death-1 (PD-1), high T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), high NK group 2 member A (NKG2A), but low tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), low interferon-gamma and low granzyme B. In addition, Sirt2 was also increased in NK cells after induction of exhaustion in vitro. Lentivirus-mediated Sirt2 silencing did not affect the acute activation and cytotoxicity of non-exhausted NK cells. However, Sirt2 silencing partially restored the expression of interferon-gamma, granzyme B and CD107a in exhausted NK cells. Meanwhile, Sirt2 silencing down-regulated TIM3, LAG3, TIGIT and NKG2A while up-regulated TRAIL on exhausted NK cells. Consequently, Sirt2 silencing restored the cytotoxicity of exhausted NK cells. Moreover, Sirt2 silencing partially ameliorates the defects in glycolysis and mitochondrial respiration of exhausted NK cells, as evidenced by increases in glycolytic capacity, glycolytic reserve, basal respiration, maximal respiration and spare respiration capacity. Accordingly, Sirt2 negatively regulates the tumoricidal activity of exhausted NK cells in CRC.

Dying tumor cells-inspired vaccine for boosting humoral and cellular immunity against cancer.

Whole tumor cells can act as effective antigen depots and have been regarded as prospective candidate for cancer vaccines. However, the clinical outcomes of whole tumor cell vaccine were restricted by the poor immunogenicity and potential in vivo oncogenicity risks. Herein, a simple and effective cancer vaccine named frozen dying tumor cells (FDT) was constructed to initiate a cascade of immune attacks against cancer. By introducing immunogenic dying tumor cells and integrating cryogenic freezing technology, FDT was endowed with high immunogenicity, good in vivo safety, and long-time storage superiority. In syngeneic mice with malignant melanoma, FDT primed the polarization of follicular helper T cells and the differentiation of germinal center B cells in lymph nodes, and promoted the infiltration of cytotoxic CD8+ T cells in the tumor microenvironment, triggering the dual activation of humoral and cellular immunity. Of note, when combined with cytokines and immune checkpoint inhibitors, the FDT vaccine achieved 100% eradication of pre-existing tumors in mice, as demonstrated in the peritoneal metastasis model of colorectal carcinoma. Taken together, our work suggests an efficient cancer vaccine inspired by dying tumor cells and provides an alternative treatment candidate for cancer.

Radiofrequency Ablation–Induced Tumor Growth Is Suppressed by MicroRNA-21 Inhibition in Murine Models of Intrahepatic Colorectal Carcinoma

PurposeTo investigate the role of microRNA-21 (miR21) in radiofrequency (RF) ablation–induced tumor growth and whether miR21 inhibition suppresses tumorigenesis. Material and MethodsStandardized liver RF ablation was applied to 35 C57/BL6 mice. miR21 and target proteins pSTAT3, PDCD4, and PTEN were assayed 3 hours, 24 hours, and 3 days after ablation. Next, 53 Balb/c and 44 C57BL/6 mice received Antago-miR21 or scrambled Antago-nc control, followed by intrasplenic injection of 10,000 CT26 or MC38 colorectal tumor cells, respectively. Hepatic RF ablation or sham ablation was performed 24 hours later. Metastases were quantified and tumor microvascular density (MVD) and cellular proliferation were assessed at 14 or 21 days after the procedures, respectively. ResultsRF ablation significantly increased miR21 levels in plasma and hepatic tissue at 3 and 24 hours as well as target proteins at 3 days after ablation (P < .05, all comparisons). RF ablation nearly doubled tumor growth (CT26, 2.0 SD ± 1.0 fold change [fc]; MC38, 1.9 SD ± 0.9 fc) and increased MVD (CT26, 1.9 SD ± 1.0 fc; MC38, 1.5 ± 0.5 fc) and cellular proliferation (CT26, 1.7 SD ± 0.7 fc; MC38, 1.4 SD ± 0.5 fc) compared with sham ablation (P < .05, all comparisons). RF ablation–induced tumor growth was suppressed when Antago-miR21 was administered (CT26, 1.0 SD ± 0.7 fc; MC38, 0.9 SD ± 0.4 fc) (P < .01, both comparisons). Likewise, Antago-miR21 decreased MVD (CT26, 1.0 SD ± 0.3 fc; MC38, 1.0 SD ± 0.2 fc) and cellular proliferation (CT26, 0.9 SD ± 0.3 fc; MC38, 0.8 SD ± 0.3 fc) compared with baseline (P < .05, all comparisons). ConclusionsRF ablation upregulates protumorigenic miR21, which subsequently influences downstream tumor-promoting protein pathways. This effect can potentially be suppressed by specific inhibition of miR21, rendering this microRNA a pivotal and targetable driver of tumorigenesis after hepatic thermal ablation.

Tumor loss-of-function mutations in STK11/LKB1 induce cachexia.

Cancer cachexia (CC), a wasting syndrome of muscle and adipose tissue resulting in weight loss, is observed in 50% of patients with solid tumors. Management of CC is limited by the absence of biomarkers and knowledge of molecules that drive its phenotype. To identify such molecules, we injected 54 human non-small cell lung cancer (NSCLC) lines into immunodeficient mice, 17 of which produced an unambiguous phenotype of cachexia or non-cachexia. Whole-exome sequencing revealed that 8 of 10 cachexia lines, but none of the non-cachexia lines, possessed mutations in serine/threonine kinase 11 (STK11/LKB1), a regulator of nutrient sensor AMPK. Silencing of STK11/LKB1 in human NSCLC and murine colorectal carcinoma lines conferred a cachexia phenotype after cell transplantation into immunodeficient (human NSCLC) and immunocompetent (murine colorectal carcinoma) models. This host wasting was associated with an alteration in the immune cell repertoire of the tumor microenvironments that led to increases in local mRNA expression and serum levels of CC-associated cytokines. Mutational analysis of circulating tumor DNA from patients with NSCLC identified 89% concordance between STK11/LKB1 mutations and weight loss at cancer diagnosis. The current data provide evidence that tumor STK11/LKB1 loss of function is a driver of CC, simultaneously serving as a genetic biomarker for this wasting syndrome.

Abstract 4038: Discovery of potent and selective inhibitors of the protein tyrosine phosphatases PTPN2 and PTPN1 to trigger anti-tumor immunity through sensitization of tumor cells and activation of immune cells

Abstract Protein tyrosine phosphatase nonreceptor type 2 (PTPN2) has emerged as a promising cancer immunotherapy target. Previously, we reported on the functional role of PTPN2 in the pathogenesis of colorectal carcinoma (CRC). We demonstrated increased PTPN2 phosphatase activity correlates with disease progression and decreased immune responses in tumor tissues, while loss of PTPN2 in T-cells or in dendritic cells (DCs) reduces tumor burden in several CRC models and potentiates anti-PD1 efficacy (Katkeviciute et al., 2021). Others reported that loss of PTPN2 in tumor cells sensitizes to immune-mediated tumor killing (Manguso et al., 2017; Goh et al., 2022). Thus, PTPN2 was proposed as a critical node to modulate anti-tumor immunity, and PTPN2 inhibition is expected to enhance anti-tumor immunity by sensitizing tumor cells and by activating immune cells. Recently, also the closely related phosphatase PTPN1 (PTP1B) was demonstrated to restrain T-cell mediated tumor killing (Wiede et al., 2022). Inhibition of Protein Tyrosine Phosphatases has historically been a demanding area for drug discovery. However, both allosteric PTP inhibitors (eg targeting SHP2/PTPN11) and catalytic-site inhibitors (targeting PTPN2/N1) have recently progressed to clinical studies. We now identified a chemical series of novel dual PTPN2/N1 inhibitors. These inhibitors bind to the catalytic site of PTPN2, as confirmed by crystallography. The best molecules of the series are low nM inhibitors of PTPN2 and PTPN1 enzymes with excellent selectivity across other phosphatases. As an immediate consequence of PTPN2/N1 inhibition, these compounds augment phosphorylation of STAT1 and/or STAT5 isoforms in myeloid and T-cell lines, and in primary T-cells, macrophages and DCs. This results in immune-cell activation as evidenced by increased production of effector cytokines (e.g. IFNγ), immune-activation, levels of cytotoxicity markers in T-cells (e.g. granzyme B) as well as upregulation of antigen presentation and co-stimulatory markers in macrophages and DCs. Furthermore, inhibition of PTPN2/N1 sensitizes murine and human cancer cell lines to IFNγ, and enhances immune-mediated tumor killing in co-culture assays in vitro. In vivo evaluation of selected examples is in progress and results will be presented at the conference. Our data indicate that inhibition of PTPN2/N1 phosphatase activity is a powerful pharmacologic strategy to promote anti-tumor immunity, with strong potential to be exploited for cancer immunotherapy both as a single agent treatment in anti-PD1 refractory cancers, and in combination with anti-PD1 therapy. Citation Format: Kalliopi Pervolaraki, Egle Katkeviciute, Dominique Lambin, Sandro Boland, Amuri Kilonda, Vincent Pericolle, Marnik Nijs, Wanda Haeck, Kristine Metzger, Hugo Klaassen, Arnaud Marchand, Patrick Chaltin, Matthias Versele, Marianne Spalinger, Michael Scharl. Discovery of potent and selective inhibitors of the protein tyrosine phosphatases PTPN2 and PTPN1 to trigger anti-tumor immunity through sensitization of tumor cells and activation of immune cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4038.