Colon Carcinoma Cells Research Articles

The genetic interaction map of the human solute carrier superfamily.

Solute carriers (SLCs), the largest superfamily of transporter proteins in humans with about 450 members, control the movement of molecules across membranes. A typical human cell expresses over 200 different SLCs, yet their collective influence on cell phenotypes is not well understood due to overlapping substrate specificities and expression patterns. To address this, we performed systematic pairwise gene double knockouts using CRISPR-Cas12a and -Cas9 in human colon carcinoma cells. A total of 1,088,605 guide combinations were used to interrogate 35,421 SLC-SLC and SLC-enzyme double knockout combinations across multiple growth conditions, uncovering 1236 genetic interactions with a growth phenotype. Further exploration of an interaction between the mitochondrial citrate/malate exchanger SLC25A1 and the zinc transporter SLC39A1 revealed an unexpected role for SLC39A1 in metabolic reprogramming and anti-apoptotic signaling. This full-scale genetic interaction map of human SLC transporters is the backbone for understanding the intricate functional network of SLCs in cellular systems and generates hypotheses for pharmacological target exploitation in cancer and other diseases. The results are available at https://re-solute.eu/resources/dashboards/genomics/ .

Demethylated miR-184 regulates EPB41L5 and downregulates Notch signaling to inhibit metastasis in colorectal cancer.

Colorectal cancer (CRC) remains a leading cause of cancer-related mortality worldwide, with metastasis being a major contributor to poor prognosis. MicroRNA-184 (miR-184) has been implicated in the progression of various cancers, but its role in CRC metastasis remains poorly defined. This study investigated the effects of miR-184 promoter demethylation on EPB41L5 expression and Notch signaling in CRC. SW620 human colon carcinoma cells were treated with the DNA methylation inhibitor 5-Aza for 96h. Methylation status was assessed via bisulfite sequencing, and gene expression was evaluated using qRT-PCR and Western blotting. Functional assays were conducted to assess cell proliferation, apoptosis, migration, and invasion. 5-Aza treatment significantly decreased miR-184 promoter methylation, leading to increased miR-184 expression. This upregulation suppressed CRC cell migration and invasion, induced G2/M cell cycle arrest, and promoted apoptosis. Mechanistically, miR-184 inhibited EPB41L5 expression, thereby downregulating the Notch signaling pathway and modulating epithelial-mesenchymal transition markers. High EPB41L5 expression in CRC tissues was associated with worse prognosis. These findings suggest that demethylated miR-184 inhibits CRC metastasis by targeting the EPB41L5/Notch signaling axis. This regulatory pathway may serve as a novel prognostic biomarker and therapeutic target, with potential clinical implications for the prevention and treatment of metastatic colorectal cancer.

Enhancing the Nutritional and Health-Related Properties of Taralli Through the Use of Pleurotus eryngii: Focus on Antioxidant and Anti-Inflammatory Properties

In this study, a portion of whole durum wheat semolina was replaced with Pleurotus eryngii powder (PeP) at concentrations of 5% and 10% (w/w) to produce two taralli variants, TPE5 and TPE10. The impact of PeP on the technological, chemical, physical, and sensory properties of taralli was evaluated. The functional characteristics of the enriched taralli were assessed employing HCT8 human colon carcinoma cells as the experimental model. The inclusion of PeP in taralli increased total dietary fiber, meeting the “high fiber” criteria under Regulation 1924/2006 while also enhancing the total phenol content. The higher fiber and polyphenol content in the enriched samples contributed to a significant reduction in glycemic index and starch hydrolysis. Treatment with PeP-enriched taralli resulted in a notable decrease in intracellular ROS levels in HCT8 cells, demonstrating strong antioxidant potential. Furthermore, TPE5 exerted beneficial effects by reducing inflammation—evidenced by a significant decrease in NFkB phosphorylation at serine 536—and by promoting apoptosis. These effects are likely mediated through the regulation of intracellular oxidative states. Overall, these findings indicate that PeP enrichment enhances the nutritional profile of taralli and provides potential health benefits, reinforcing its role as a valuable functional ingredient.

Unraveling the interaction of pyranocoumarins with P-glycoprotein: Implications for overcoming multidrug resistance in cancer therapy.

Unraveling the interaction of pyranocoumarins with P-glycoprotein: Implications for overcoming multidrug resistance in cancer therapy.

Arctic lichen Cladonia borealis-induced cell death is mediated by p53-independent activation of Caspase-9 and PARP-1 signaling in human colorectal cancer cell lines

The anti-cancer effects of Cladonia borealis (an Arctic lichen) methanol extract (CBME) on human colon carcinoma HCT116 cells were investigated for the first time. The proliferation of the HCT116 cells treated with CBME significantly decreased in a dose- and time-dependent manner. Flow cytometry results indicated that treatment with CBME resulted in significant apoptosis in the HCT116 cells. Furthermore, immunoblotting and qRT-PCR results revealed the expression of apoptosis-related marker genes and indicated a significant downregulation of the apoptosis regulator B-cell lymphoma expression and upregulation of the cleaved form of poly (ADP-ribose) polymerase as DNA repair and apoptosis regulators and central tumor suppressor p53. Therefore, CBME significantly inhibited cell proliferation by inducing apoptosis via the mitochondrial apoptotic pathway in colon carcinoma cells. Collectively, these data suggested that CBME contained one or more compounds with anti-cancer effects and could be a potential therapeutic agent. Further studies are required to identify candidate compounds and understand the mechanism of action of CBME.

Abstract 2536: In vivo label-free imaging reveals distinct CD8 T cell metabolic changes during effective radio-immunotherapy in cold murine melanoma versus hot colon carcinoma

Abstract In vivo multiphoton autofluorescence microscopy provides label-free, single-cell imaging of metabolic changes. These metabolic changes are quantified via the metabolic coenzymes NAD(P)H and FAD which are autofluorescent molecules endogenous to all cells. Metabolic reprogramming is a hallmark of cancer. Immune cells also change their metabolism during cancer progression and this metabolic switch correlates to immune cell phenotype and function. Here, we study metabolic changes during administration of a potent radio-immunotherapy regimen in murine melanoma and colon carcinoma. This therapy includes external beam radiation, hu14.18-IL2 immunocytokine (anti-GD2 mAb fused to IL2) or free IL2, and anti-CTLA-4, leading to in situ vaccination and cure of established murine tumors. We created a transgenic mCherry-labeled T cell mouse to study the critical T cell response. We implanted syngeneic B78 (GD2+) melanoma or MC38 (GD2-) colon carcinoma cells into the flanks of mCherry-labeled CD8 T cell reporter mice to induce tumors. Under anesthesia, skin flap surgery was performed, and tumors were imaged at multiple timepoints during therapy. Multiphoton imaging was performed to collect NAD(P)H, FAD, and mCherry signal. Fluorescence lifetime images were collected using time correlated single photon counting electronics. Tissues were also harvested and analyzed via flow cytometry, single-cell RNA sequencing, and multiplex immunofluorescence to corroborate imaging findings and characterize the immune infiltrate. Our in vivo label-free imaging revealed that CD8 T cells from treated versus control tumors exhibit different metabolic phenotypes including changes in NAD(P)H and FAD protein binding and redox balance across these two solid tumors. In cold B78 melanoma tumors, in vivo metabolic imaging revealed that CD8 T cells are activated early but maintain an oxidative metabolic state consistent with exhaustion or a memory phenotype. In hot MC38 colon carcinoma, in vivo metabolic imaging revealed that CD8 T cells are activated early and then quickly transition to a glycolytic state that fuels their cytotoxic function. scRNAseq confirmed the metabolic changes we observed within our B78 model and provided additional insight into CD8 T and tumor cell changes during therapy. Flow cytometry illustrated significant changes in the immune infiltrates of both tumor types over time and treatment. These results show that label-free in vivo imaging enables single-cell quantification of metabolic changes during therapy - across multiple solid tumors. Combined with other traditional assays, we can elucidate key immune cell populations and the crucial timepoints during therapy where changes are occurring. This imaging platform may be leveraged to help develop new immunotherapy combinations and study other cancer types where cures have not been realized. Citation Format: Alexa R. Heaton, Anna Hoefges, Nathaniel J. Burkard, Arika S. Feils, Noah W. Tsarovsky, Garrett M. Lublin, Alina A. Hampton, Samantha K. Burkard, Aurora D'Amato, Mildred A. Felder, Anqi Gao, Huy Q. Dinh, Amy K. Erbe-Gurel, Alexander L. Rakhmilevich, Paul M. Sondel, Melissa C. Skala. In vivo label-free imaging reveals distinct CD8 T cell metabolic changes during effective radio-immunotherapy in cold murine melanoma versus hot colon carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2536.

Abstract 6061: Dual PD-L1 blockade and VEGF-A neutralization with the bispecific antibody BNT327/PM8002 shows potent antitumor activity in preclinical models

Abstract The combined blockade of the PD-1/PD-L1 checkpoint and VEGF-A driven angiogenesis has been shown to reduce immune suppression in the tumor microenvironment, to enhance antitumor immune responses, and exhibit improved antitumor activity compared to each individual treatment. BNT327 (also known as PM8002) is an investigational bispecific antibody (bsAb) composed of a bivalent Fc-silenced anti-VEGF-A IgG1 antibody fused at its C-terminus to two humanized anti-PD-L1 single-domain VHH antibodies. By simultaneously targeting PD-L1 and VEGF-A, BNT327 is designed to combine the two complementary functions of amplifying pre-existing immune responses and anti-angiogenesis. Here, we report preclinical studies characterizing the functional activity of BNT327 in vitro and antitumor activity in vivo. BNT327 bound with high affinity (i.e., nanomolar or subnanomolar KD) to recombinant human PD-L1 and VEGF-A, as shown by biolayer interferometry. In cell-based bioluminescent reporter assays, BNT327 inhibited PD-L1/PD-1 signaling and VEGF-A/VEGFR2 signaling with subnanomolar EC50 values. In mixed lymphocyte reaction assays using human PBMCs and allogeneic monocyte-derived dendritic cells, BNT327 and the parental anti-PD-L1 VHH dose-dependently increased IL-2 and IFN-γ secretion. Furthermore, BNT327 dose-dependently enhanced CD8+ T-cell mediated tumor-cell killing in an antigen-specific cytotoxicity assay in vitro, with the effects being comparable to those of PD-L1 blockade by atezolizumab. In vivo, a BNT327 surrogate bsAb targeting human PD-L1 and mouse VEGF-A exhibited dose-dependent antitumor activity in human PD-L1 knock-in mice bearing syngeneic human PD-L1-expressing CT26 tumors. Tumor growth inhibition was superior to individual treatments with PD-L1 blockade (parental anti-PD-L1 or atezolizumab) or with anti-VEGF-A. Furthermore, BNT327 showed antitumor activity in several xenograft models, including A375 melanoma cells, NCI-H1975 lung carcinoma cells, LoVo colon carcinoma cells, and MDA-MB-231 breast carcinoma cells. Antitumor activity was dose-dependent where tested, and superior to single PD-1 (pembrolizumab) or PD-L1 (atezolizumab) checkpoint inhibitor treatment. In conclusion, BNT327 inhibits PD-1/PD-L1 signaling and VEGF-A/VEGFR2 signaling in vitro and shows potent antitumor activity in vivo both in syngeneic and xenograft models. The safety and efficacy of BNT327 as a monotherapy or in combination is being investigated in multiple clinical trials in patients with solid tumors, including Phase II and Phase III trials investigating the combination with chemotherapeutic agents in triple-negative breast cancer and lung cancer. Citation Format: Xiaoniu Miao, Yifeng Xu, Shaogang Peng, Weifeng Huang, Junying Chen, Maren Köhne, Andrea Imle, Alexander Muik, Özlem Türeci, Ugur Sahin, Andy Tsun, Yi Luo. Dual PD-L1 blockade and VEGF-A neutralization with the bispecific antibody BNT327/PM8002 shows potent antitumor activity in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6061.

Abstract 639: Targeting TET3 potentiates an effective anti-tumor immune response after irradiation

Abstract The benefits of radiotherapy (RT) have long been appreciated as limited by formation and repair of DNA double-strand breaks (DSBs) in tumor cells, but recent understanding has identified a key role for RT and DNA damage in promoting inflammation that drives anti-tumor immune response. While chromosomal DSBs indeed directly limit cancer cell survival and proliferation, RT-induced damage leads to cytosolic DNA that activates cGAS/STING/IRF3 signaling and drives IFNα/β expression, recruiting cytotoxic T and NK cells to shrink tumors. In turn, along with DSB repair, another determinant of RT resistance is rebound immunosuppression. Here, IFNγ released by the infiltrating lymphocytes activates JAK/STAT to induce interferon-stimulated genes (ISGs) including PD-L1 that can reduce tumor inflammation and restore growth. Though combining PD-1/PD-L1 blockade with RT is sufficient to block this adaptive response in mice, the lack of benefit to patients suggests the need to more broadly target immunosuppressive ISG expression. Targeting IFNγ signaling by blocking JAK/STAT may limit immunosuppressive ISG expression but will impair T and NK cytotoxicity. Looking downstream, blocking JAK/STAT-dependent ISG promoter CpG demethylation by Ten Eleven Translocation (TET) dioxygenase enzymes may prevent PD-L1 expression without affecting cytotoxicity. In this study, we disrupted TET activity with small-molecule inhibitors and CRISPR RNP knockouts (KO) to evaluate effects on radiation response in vitro and in tumors. As predicted, TET inhibitors blocked PD-L1 induction in irradiated CT26 murine colon carcinoma cells. Assaying TET1, 2 and/or 3 KO CT26 cells for PD-L1 induction identified TET3 as the critical mediator. TET3 KO cells also exhibited slower DSB repair after irradiation, associated with markedly increased ssDNA resection and cytosolic DNA. The irradiated TET3 KO cells also displayed enhanced IFNα/β expression, suggesting intact cGAS/STING/IRF3 signaling. While TET3 KO CT26 cells formed tumors in syngeneic BALB/c mice much like CT26 controls, RT produced greater NK cell infiltrates by one week and increased tumor elimination by two weeks. TET3 KO 4T1 mammary carcinoma cells formed orthotopic tumors in BALB/c mice but produced fewer lung metastases compared to controls. In conclusion, TET3 may offer an attractive, druggable target for radiosensitization by slowing DSB repair, enhancing tumor inflammation, and blocking immunosuppressive ISG expression. Citation Format: Sera Averbek, Stephen J. Kron. Targeting TET3 potentiates an effective anti-tumor immune response after irradiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 639.

Colon cancer exosome-associated HSP90B1 initiates pre-metastatic niche formation in the liver by polarizing M1 macrophage into M2 phenotype

BackgroundColorectal cancer (CRC) frequently metastasizes to the liver, worsening patient outcomes. The formation of a pre-metastatic niche (PMN) is essential for this process, but how the primary colon tumor orchestrates the PMN formation remains unclear.MethodsThis study investigated the role of CRC-derived exosomes using CT-26 murine colon carcinoma cells. The effects of these exosomes on immune cells, specifically M1 macrophage polarization and CD8 + T cell viability, were assessed. HSP90B1 expression in CT-26-derived exosomes was analyzed to understand its contribution to PMN formation. HSP90B1 silencing experiments were conducted to evaluate its impact on immunosuppressive PMN creation and liver metastasis. Patient blood samples were also examined to correlate exosomal HSP90B1 levels with CRC progression.ResultsExosomes from CT-26 cells were found to polarize M1 macrophages into an M2 phenotype and decrease CD8 + T cell viability, promoting liver metastasis. High expression of HSP90B1 in CT-26 cell-derived exosomes was identified as a key factor in inducing M2 macrophage polarization and creating an immunosuppressive PMN. Silencing HSP90B1 significantly inhibited the exosome-mediated formation of the immunosuppressive PMN and reduced liver metastasis. Furthermore, elevated levels of HSP90B1 in patient-derived exosomes were associated with advanced CRC and poorer prognosis.ConclusionsCRC-derived exosomes promote liver metastasis by forming an immunosuppressive PMN through HSP90B1. Targeting HSP90B1 in CRC exosomes may offer a new therapeutic strategy to prevent liver metastasis and improve patient outcomes.

Antiviral Effect of Erdosteine in Cells Infected with Human Respiratory Viruses.

Respiratory viral infections trigger immune and inflammatory responses that can be associated with excessive oxidative stress, glutathione (GSH) depletion, and a cytokine storm that drives virus-induced cell/tissue damage and severe disease. Erdosteine is a thiol-based drug with proven mucolytic, anti-inflammatory, antioxidant, and antibacterial properties, but less is known about its antiviral effects. We performed in vitro studies to investigate the antiviral and anti-inflammatory activity of erdosteine in A549-hACE2 human lung epithelial cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or respiratory syncytial virus (RSV) and in Caco-2 human colon carcinoma cells infected with influenza A virus (H1N1). The cells were treated with different concentrations of erdosteine or its active metabolite 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MET-1) before and after viral infection. The viral replication/load in the cell culture supernatants was measured by real-time quantitative polymerase chain reaction (RT-qPCR) assay and digital droplet PCR. The gene expression of innate immune response signaling pathways and oxidative stress was analyzed by reverse transcription PCR custom-array. The results showed that erdosteine and its active metabolite, at concentrations consistent with an approved therapeutic human dosage, were not directly cytotoxic and had significant antiviral effects in cells pre-infected with SARS-CoV-2, RSV, and H1N1. The transcriptome analysis showed that erdosteine activated innate immune responses by stimulating overexpression of type I interferon and inflammasome pathways and modulated oxidative stress by inducing the modulation of oxidative stress and GSH pathways. These findings suggest that erdosteine may be a useful treatment for respiratory viral infections.

Perfluorohexane Emulsions Induce Macrophage-Stimulating Effects through iNOS Expression.

Despite advances in cancer immunotherapy, efficacy has been limited to a subset of patients, highlighting the need for continued development of innovative therapeutic approaches. Perfluorocarbon-filled (PFC) particles, due to unique physicochemical properties, biological stability, and quantifiability as 19F-MRI tracers, have been intensely investigated as in vivo macrophage imaging agents. Despite this, little is known about the effect of PFC particle uptake on macrophage behavior. Here, phospholipid-stabilized perfluorohexane (PFH) nanodroplets (NDs) were used to treat bone-marrow-derived macrophages (BMDMs) in vitro and tumor-implanted mice in vivo. Physiological response was assessed by using biochemistry, flow cytometry, fluorescence imaging, and tumor measurements. The incubation of alternatively-activated (M2) BMDMs with PFH NDs led to a dose- and time-dependent increase of iNOS expression but not that of other common M1 or M2 markers. PFH ND-induced iNOS expression was further enhanced when treated M2 BMDMs were co-cultured with MC38 murine colon carcinoma cells, resulting in more effective tumoral cell apoptosis. Similarly, intratumoral injection of PFH NDs in a murine colon carcinoma model led to increased iNOS expression within the tumors and caused a substantial decrease in tumor size. This size reduction was also unexpectedly accompanied by the formation of peritumoral gas pockets, which, when aspirated, abrogated tumoral effects. Conversely, intratumoral injection of emulsions made with high-boiling-point (BP = 142 °C) perfluorooctyl bromide (PFOB) did not produce gas pockets or inhibit tumor growth. Our data collectively show the immunogenic potential of PFH NDs through iNOS expression and promote the potential development of theranostic PFC-based platforms.

Dual role of p21 in regulating apoptosis and mitotic integrity in response to doxorubicin in colon cancer cells

This study explores the multifaceted role of p21 in mediating cellular responses to DNA-damaging agents, with a focus on doxorubicin treatment in HCT116 colon carcinoma cells. We investigated how different doses of doxorubicin affect cells with varied p21 and p53 statuses, revealing distinct roles for p21 depending on the drug dosage. At high doses (HD), p21 is more critical than p53 in mediating apoptosis, whereas at low doses (LD), p21 is essential for preventing mitotic defects and multinucleation. Notably, reintroducing p21 or pharmacologically inhibiting CDK1 reduced multinucleation. The absence of p21 upon LD doxorubicin exposure led to aberrant chromosome segregation, persistent DNA damage response (DDR) activation, and increased non-homologous end-joining (NHEJ) activity, resulting in unrepaired DNA accumulation and multinucleation. Additionally, mitotic defects in p21-deficient cells were associated with mislocalization of key mitotic regulators, Aurora B and mitotic kinesin-like protein 1 (MKLP1), exacerbating defective mitosis. In summary, p21 functions as a dual regulator in response to DNA damage, promoting apoptosis at HD and preventing mitotic failure at LD. These insights have significant implications for cancer therapy, highlighting the potential of targeting the p21 to enhance treatment efficacy.

Comparative analysis of Caco-2 cells and human jejunal and duodenal enteroid-derived cells in gel- and membrane-based barrier models of intestinal permeability.

Comparative analysis of Caco-2 cells and human jejunal and duodenal enteroid-derived cells in gel- and membrane-based barrier models of intestinal permeability.

Enhancing efficacy of combretastatin A4 by encapsulation in solid lipid nanoparticles: Implications for anti-angiogenic cancer therapy.

Enhancing efficacy of combretastatin A4 by encapsulation in solid lipid nanoparticles: Implications for anti-angiogenic cancer therapy.

MTT assay-based evaluation of live, heat-killed, and cell-free supernatants of probiotic bacteria on CT-26 murine colon carcinoma cells

MTT assay-based evaluation of live, heat-killed, and cell-free supernatants of probiotic bacteria on CT-26 murine colon carcinoma cells

Symmetrical di-substituted phenylamino-s-triazine derivatives as anticancer agents: in vitro and in silico approach.

A series of symmetrical tri-substituted s-triazine derivatives were designed and synthesized by two different methods (reflux and microwave-assisted methods). The structures of compounds were determined by infrared (IR), nuclear magnetic resonance (1H NMR and 13C NMR), and mass spectrometry. The yield of the microwave-assisted method (91-98%) was significantly higher (about 10%) than that of the reflux method (80-88%) meanwhile the reaction time was significantly shorter (15-30 min). Compound 3b showed good cytotoxic activity against the MCF7 (human breast cancer) cell line with an IC50 value of 6.19 μM. Compounds 3a and 2e showed strong cytotoxic activity against the C26 (colon carcinoma) cell line with IC50 values of 1.21 and 8.28 μM, respectively. Compound 3e showed good cytotoxic activity against both MCF7 and C26 cell lines with IC50 values of 13.74, and 14.66 μM respectively. In particular, compound 2d exhibited the best potent cytotoxic activity among the synthesized compounds against both MCF7 and C26 cell lines with IC50 values of 6.54 and 0.38 μM, respectively. Moreover, compounds 2e, 3a, and 3e showed higher selectivity on cancer cell lines and lower toxicity on BAEC (bovine aorta endothelial) normal cells compared to compounds 2d, 3a, paclitaxel, and doxorubicin. In silico studies revealed five potent compounds with good physicochemical and ADMET profiles and potent interactions with key anticancer targets (EGFR, DHFR, VEGFR2, CDK2, mTOR, and PI3K) compared to reference drugs. This work paved the way for the synthesis of more potent compounds based on the phenylamino-s-triazine scaffold and the exploration of their diverse and potential biological activities as well as their mechanisms of action.

Induced clustering of SHP2-depleted tumor cells in vascular islands restores sensitivity to MEK/ERK inhibition.

Allosteric inhibitors of the tyrosine phosphatase SHP2 hold therapeutic promise in cancers with overactive RAS/ERK signaling but "adaptive resistance" to SHP2 inhibitors may limit benefits. Here, we utilized tumor cells that proliferate similarly with or without endogenous SHP2 to explore means to overcome this growth-independence from SHP2. We found that SHP2 depletion profoundly alters output of vascular regulators, cytokines, chemokines, and other factors from SHP2 growth-resistant cancer cells. Tumors derived from inoculation of SHP2-depleted, but SHP2 growth-independent, mouse melanoma and colon carcinoma cell lines display a typically subverted architecture where proliferative tumor cells cluster in distinct "vascular islands" centered by remodeled vessels, each limited by surrounding hypoxic and dead tumor tissue, where inflammatory blood cells are limited. Although vascular islands generally reflect protected sanctuaries for tumor cells, we found that vascular island-resident, highly proliferative, SHP2-depleted tumor cells acquire an increased sensitivity to blocking MEK/ERK signaling resulting in reduced tumor growth. Our results show that response to targeted therapies in resistant tumor cells is controlled by tumor cell-induced vascular changes and tumor architectural reorganization providing a compelling approach to eliciting tumor response by exploiting tumor and endothelial-dependent biochemical changes.

Inhibition of dendritic cell maturation in 3D and 2D colon carcinoma cell models

Dendritic cells (DCs) play an important role in the immune response against cancer. Numerous studies indicate that tumor microenvironment affects the phenotype and function of DCs. The aim of the work was to determine how three-dimensional (3D) versus two-dimensional (2D) mixed-cells tumor models (mouse CT26 colon cancer cells mixed with mouse 3T3 fibroblasts) can impact on DCs maturation. Three-days old spheroids of 200μm diameter were used, formed by mixing CT26 and 3T3 cells cultivated in 3D Petri DishTM (microtissuesTM) molds. DCs were isolated from BALB/c mouse femurs and added to mixed-cell 3D spheroids and 2D mixed-cell monolayer, in coculture. DCs control group and Wnt-inhibited DCs (by XAV939) were also used. After 48 hours of coculture, half of the DCs were collected and analyzed by flow cytometry. Splenocytes were added to the remaining half of the DCs, cocultured for another 48 hours, and T-cell activation analysis was performed by flow cytometry. Here we show that mixed colorectal carcinoma spheroids have significantly higher immunoinhibitory properties than 2D mixed cell culture. Inhibition of Wnt signaling pathway doesn't affect the maturation of DCs but decreases the expression of checkpoint molecules (PD-1, Tim3) on CD4+ T-cells primed by XAV939-treated DCs. Acknowledgments: Institutional Grant RVO 61388971 (CZ), The National Institute for Cancer Research Project (EXCELES, ID Project No. LX22NPO5102) funded by the European Union - Next Generation EU, the Ministry of Health of the Czech Republic project NU23-08-00071; the Generali/Česká pojišťovna a.s. (CZ), and UniCredit Bank CZ&SK a.s., Praha (CZ) sponsorships.

Antitumor efficacy of intermittent low-dose erlotinib plus sulindac via MHC upregulation and remodeling of the immune cell niche.

A previously reported clinical trial in familial adenomatous polyposis (FAP) patients treated with erlotinib plus sulindac (ERL + SUL) highlighted immune response/interferon-γ signaling as a key pathway. In this study, we combine intermittent low-dose ERL ± SUL treatment in the polyposis in rat colon (Pirc) model with mechanistic studies on tumor-associated immune modulation. At clinically relevant doses, short-term (16 weeks) and long-term (46 weeks) ERL ± SUL administration results in near-complete tumor suppression in Pirc colon and duodenum (p < 0.0001). We identify a low-dose threshold for significant antitumor activity in Pirc rats given SUL at 125 ppm in the diet plus ERL at 5 mg/kg body weight via twice-weekly oral gavage (SUL125 + ERL5 × 2). Longitudinal analyses show diminished expression of MHC class I and II genes in polyps larger than Grade 5, a novel finding in the Pirc model. Treatment with ERL ± SUL upregulates the corresponding MHC and immune-associated factors in a subset of Pirc colon polyps, Pirc tumor cell lines, murine colon carcinoma cells, and FAP patient-derived organoids, with Nlrc5 playing a critical role in this effect. Imaging mass cytometry reveals that SUL125 + ERL5 × 2 increases tumor-associated Cd4+ T cells by ~2.6-fold (p < 0.05), with no apparent effect on Cd8+ T cells. The treatment also increases tumor-associated Cd68+ cells (p < 0.05) and decreases Foxp3+ (p < 0.01) and Arg1+ (p < 0.05) cells. Thus, intermittent low-dose ERL + SUL treatment enhances tumor-associated MHC expression and remodels the immune cell niche toward a more permissive "helper" immune microenvironment. We conclude that early immune-interception strategies targeting interferon-γ signaling may benefit FAP patients at drug doses below the clinical standard of care.

Metabolite Profiling and Toxicity, Antioxidant, and Antitumor Evaluation of Micromeria biflora Aerial Parts Extract Combined with ADMET Prediction and Molecular Docking Analysis.

Micromeria biflora is widely used traditionally for various disorders, such as wounds, nosebleeds, and sinusitis. The study was conducted to investigate the chemical and pharmacological properties of M. biflora aerial parts. Gas chromatography-mass spectrometry (GC-MS) analysis revealed the presence of 84 components in M. biflora total methanolic extract (MBTME). The toxicity study showed no morbidity and/or mortality at the tested concentrations. The estimated LD50 value of MBTME was greater than 3g/kg body weight. MBTME exhibited significant 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and increased reducing power in the ferric reducing antioxidant power (FRAP) assay with the elevation in concentration. MBTME demonstrated significant anticancer efficacy against hepatocellular carcinoma (HepG-2), colon carcinoma (HCT-116), and intestinal carcinoma (Caco-2) cell lines with IC50 values of 23.14 ±2.92, 28.22 ±3.07, and 18.70 ±3.36 µg/mL, respectively. Histological examination revealed substantial morphological changes consistent with the observation associated with the apoptotic mechanism of action. The molecular docking study provided insights into the rational binding modes of the identified compounds with poly(ADP-ribose) polymerase-1 (PARP-1) and tyrosinase. In silico absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties predictions found that most of the identified compounds exhibited low toxic effects and good absorption and solubility properties.