Orthotopic Colorectal Cancer Model Research Articles

Abstract 1386: CD1d-restricted iNKT cells promote liver metastasis from colon cancer

Abstract The liver is a prominent site for metastasis, yet little is known about the role of liver immunity in antimetastatic defense. Compared to other organs, the liver harbors a unique immune environment characterized by the abundance of innate cell lineages, including CD1d-restricted invariant natural killer T (iNKT) cells. To investigate the influence of iNKT cells on metastasis, we established an orthotopic model of colorectal cancer (CRC) with spontaneous dissemination to the liver. We induce the primary tumor by implanting CRC organoids into the colon mucosa of syngeneic C57BL/6 mice, resulting in hepatic macrometastases within 5 weeks. We observed that liver metastasis hardly occurred in mouse strains lacking iNKT cells (Cd1d−/− and Traj18−/− mice), while the growth of the primary tumor was comparable to that in wild-type mice. These results indicate a tumor-promoting function for hepatic iNKT cells. We uncovered that hepatic iNKT cells respond to disseminating cancer by producing IL-4 and IL-13, two cytokines known to activate hepatic stellate cells (HSCs) into fibrogenic myofibroblasts. Indeed, we saw rapid activation of HSCs and deposition of extracellular matrix upon arrival of cancer cells in the liver of wild-type but not iNKT cell-deficient mice. We propose that hepatic iNKT cells contribute to the formation of a supportive niche for disseminated tumor cells by activating HSCs early in the development of hepatic metastasis. Taken together, our findings highlight an essential tumor-promoting role for iNKT cells during the development of liver metastasis and contribute to our understanding of immune regulation of metastasis. Citation Format: Marc Nater, Giulia Lucchiari, Michael Brügger, Tomas Valenta, Virginia Cecconi, Geo Forni, Stephan Benke, Maries van den Broek. CD1d-restricted iNKT cells promote liver metastasis from colon 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 1386.

P073 Enhancing the MFSD2A protein for the treatment of colitis-associated cancer: novel insights for pathogenesis and treatment

Abstract Background The intrinsic connection between inflammation and tumor promotion is well characterized and is a key pathogenic event in patients with colorectal cancer (CRC). A small fraction of patients with CRC suffers from genetic predisposition. However chronic inflammation, such as ulcerative colitis (UC), increases the risk of intestinal carcinogenesis, thus strengthening the connection between these conditions. A few years ago, our laboratory described that the intestinal endothelium isolated from actively inflamed UC patients displayed defective production of inflammation-resolving DHA-derived metabolites, by comparison with healthy controls and tissues in remission. We also reported that the Major Facilitator Superfamily Domain containing 2A (MFSD2A) participated in the production of the inflammation-resolving DHA-derived metabolites by the intestinal endothelium of patients with UC in remission, ameliorating colonic inflammation. Therefore, we hypothesized that the endothelial MFSD2A also has a role in preventing and/or counteracting cancer-associated inflammation. Methods Human Intestinal Microvascular Endothelial Cells (HIMEC) isolated from CRC and healthy samples were transduced with either MFSD2A protein-encoding lentiviral vectors (MFSD2A) or GFP as control, or MFSD2A targeting shRNA or its scramble, and were analyzed by lipidomics. Furthermore, Caco-2 cells co-cultured with HIMEC-MFSD2A were analyzed by FACS, evaluating their proliferation. Moreover, an orthotopic model of CRC was made intrarectally injecting CD1 nude mice with a cell mixture of Caco-2 and HUVEC overexpressing MFSD2A or GFP as control. Mice were gavaged with DHA-ethyl-ester or PBS. FACS analysis was performed on tumor and colonic samples. Results Lipidomic analysis revealed that the loss of MFSD2A in CRC is detrimental because there is a reduced production of pro-resolving lipids confirming that, as for UC, MFSD2A is important for the maintenance of an adequate balance between pro-resolving and pro-inflammatory phenotype. Moreover, enhancing MFSD2A expression at endothelial level limits Caco-2 cell proliferation, supporting its beneficial role. In addition, DHA administration in mice ameliorated clinical symptoms of inflammation upon intestinal endothelial MFSD2A overexpression, supporting the beneficial role of MFSD2A in vivo. Conclusion Tumor-associated inflammation remains a crucial hallmark of CRC. Our study seeks to identify the role of MFSD2A in the resolution phase of inflammation, pointing out alternative therapies for CRC treatment. These data suggested that MFSD2A might contrast the tumor-associated inflammation and ensure the correct balance between pro-inflammatory and pro-resolving milieu.

Centrosomal Protein 55 (CEP55) Drives Immune Exclusion and Resistance to Immune Checkpoint Inhibitors in Colorectal Cancer.

Colorectal cancer (CRC) currently ranks as the third most common cancer in the United States, and its incidence is on the rise, especially among younger individuals. Despite the remarkable success of immune checkpoint inhibitors (ICIs) in various cancers, most CRC patients fail to respond due to intrinsic resistance mechanisms. While microsatellite instability-high phenotypes serve as a reliable positive predictive biomarker for ICI treatment, the majority of CRC patients with microsatellite-stable (MSS) tumors remain ineligible for this therapeutic approach. In this study, we investigated the role of centrosomal protein 55 (CEP55) in shaping the tumor immune microenvironment in CRC. CEP55 is overexpressed in multiple cancer types and was shown to promote tumorigenesis by upregulating the PI3K/AKT pathway. Our data revealed that elevated CEP55 expression in CRC was associated with reduced T cell infiltration, contributing to immune exclusion. As CRC tumors progressed, CEP55 expression increased alongside sequential mutations in crucial driver genes (APC, KRAS, TP53, and SMAD4), indicating its involvement in tumor progression. CEP55 knockout significantly impaired tumor growth in vitro and in vivo, suggesting that CEP55 plays a crucial role in tumorigenesis. Furthermore, the CEP55 knockout increased CD8+ T cell infiltration and granzyme B production, indicating improved anti-tumor immunity. Additionally, we observed reduced regulatory T cell infiltration in CEP55 knockout tumors, suggesting diminished immune suppression. Most significantly, CEP55 knockout tumors demonstrated enhanced responsiveness to immune checkpoint inhibition in a clinically relevant orthotopic CRC model. Treatment with anti-PD1 significantly reduced tumor growth in CEP55 knockout tumors compared to control tumors, suggesting that inhibiting CEP55 could improve the efficacy of ICIs. Collectively, our study underscores the crucial role of CEP55 in driving immune exclusion and resistance to ICIs in CRC. Targeting CEP55 emerges as a promising therapeutic strategy to sensitize CRC to immune checkpoint inhibition, thereby improving survival outcomes for CRC patients.

Etoposide-Entrapped Progesterone-Cationic Lipid Nanoaggregates as Selective Therapeutics against Etoposide-Resistant Colorectal Cancer Cells.

Drug resistance has a major impact on the treatment of several cancers. This is mainly due to the overexpression of cellular drug efflux proteins. Hence, drug-delivery systems that can avoid this resistance are needed. We report PR10, a progesterone-cationic lipid conjugate, as a self-assembling nanoaggregate that delivers a drug cargo of etoposide, a topoisomerase inhibitor, selectively to cancer cells. In this study, we observed that etoposide nanoaggregates (P : E) caused selective and enhanced toxicity in etoposide-resistant CT26 cancer cells (IC50 9 μM) compared to when etoposide (IC50 >20 μM) was used alone. Concurrently, no toxicity was observed in etoposide-sensitive HEK293 cells for P : E treatment (IC50 >20 μM). The P : E-treated cancer cells seem to have no effect on ABCB1 expression, but etoposide-treated cells exhibited a twofold increase in ABCB1 expression, a potent efflux protein for several xenobiotic compounds. This observation supports the notion that the enhanced toxicity of P : E nanoaggregates is due to their ability to keep the expression of ABCB1 low, thus allowing longer intracellular residence of etoposide. In a BALB/c orthotopic colorectal cancer model, the nanoaggregates led to enhanced survival (45 days) compared to etoposide-treated mice (39 days). These findings suggest that PR10 could be used as a potential cancer-selective etoposide delivery vehicle to treat several etoposide-resistant cancers with fewer side effects due to the nonspecific toxicity of the drug.

Nanocatalytic bacteria disintegration reverses immunosuppression of colorectal cancer.

Tumor-associated bacteria (TAB) play a critically important role in regulating the microenvironment of a tumor, which consequently greatly deteriorates the therapeutic effects by chemo- and radiotherapy deactivation and, more considerably, leads to substantial immunosuppression. On the contrary, herein we propose a nanocatalytic tumor-immunotherapeutic modality based on the bacteria disintegration by bacteria-specific oxidative damage under magnetic hyperthermia for highly effective immune response activation-promoted tumor regression. A monodispersed and superparamagnetic nanocatalytic medicine modified by arginyl-glycyl-aspartic acid (RGD) and (3-carboxypropyl)triphenylphosphonium bromide (TPP), named as MNP-RGD-TPP herein, has been synthesized, which features selective accumulation at the TAB by the electrostatic affinity, enabling effective TAB disintegration by the nanocatalytic Fenton reaction producing abundant cytotoxic hydroxyl radicals in situ under alternating magnetic field-induced hyperthermia. More importantly, the lipopolysaccharide has been metabolically secreted from the destructed TAB as pathogen-associated molecular patterns (PAMPs) to M1-polarize tumor-associated macrophages (TAMs) and promote the maturation of dendritic cells (DCs) for innate immuno-response activation of TAMs, followed by cytotoxic T lymphocytes awakening under the PAMPs presentation by the mature DCs against tumor cells. The integrated innate and adaptive immunity activations based on this TAB-promoted nanocatalytic immunomedicine, instead of magnetic heating-induced hyperthermia or the released Fe2+/Fe3+ Fenton agent, has been found to achieve excellent therapeutic efficacy in an orthotopic colorectal cancer model, demonstrating the great potential of such an integrated immunity strategy in clinical tumor immunotherapy.

GSDMD-dependent pyroptotic induction by a multivalent CXCR4-targeted nanotoxin blocks colorectal cancer metastases

Colorectal cancer (CRC) remains the third cause of cancer-related mortality in Western countries, metastases are the main cause of death. CRC treatment remains limited by systemic toxicity and chemotherapy resistance. Therefore, nanoparticle-mediated delivery of cytotoxic agents selectively to cancer cells represents an efficient strategy to increase the therapeutic index and overcome drug resistance. We have developed the T22-PE24-H6 therapeutic protein-only nanoparticle that incorporates the exotoxin A from Pseudomonas aeruginosa to selectively target CRC cells because of its multivalent ligand display that triggers a high selectivity interaction with the CXCR4 receptor overexpressed on the surface of CRC stem cells. We here observed a CXCR4-dependent cytotoxic effect for T22-PE24-H6, which was not mediated by apoptosis, but instead capable of inducing a time-dependent and sequential activation of pyroptotic markers in CRC cells in vitro. Next, we demonstrated that repeated doses of T22-PE24-H6 inhibit tumor growth in a subcutaneous CXCR4+ CRC model, also through pyroptotic activation. Most importantly, this nanoparticle also blocked the development of lymphatic and hematogenous metastases, in a highly aggressive CXCR4+ SW1417 orthotopic CRC model, in the absence of systemic toxicity. This targeted drug delivery approach supports for the first time the clinical relevance of inducing GSDMD-dependent pyroptosis, a cell death mechanism alternative to apoptosis, in CRC models, leading to the selective elimination of CXCR4+ cancer stem cells, which are associated with resistance, metastases and anti-apoptotic upregulation.

PEGylated and Non-PEGylated TCP-1 Probes for Imaging of Colorectal Cancer.

Previous studies indicate that 99mTc- and fluorescent-labeled c[Cys-Thr-Pro-Ser-Pro-Phe-Ser-His-Cys]OH (TCP-1) peptides were able to detect colorectal cancer (CRC) and tumor-associated vasculature. This study was designed to characterize the targeting properties of PEGylated and non-PEGylated TCP-1 peptides for CRC imaging. Cell uptake of cyanine 7 (Cy7)-labeled TCP-1 probes (Cy7-PEG4-TCP-1 and Cy7-TCP-1) was investigated in three CRC cell lines (human, HCT116 and HT29; mouse, CT26). Xenograft and orthotopic CRC tumor models with HCT116 and CT26 cells were used to characterize biodistribution and CRC tumor-targeting properties of TCP-1 fluorescence and radioligand with and without PEGylation, [99mTc]Tc-HYNIC-PEG4-TCP-1 vs. [99mTc]Tc-HYNIC-TCP-1. Fluorescence images showed that TCP-1 probes were distributed in the cytoplasm and nucleus of CRC cells. When CT26 cells were treated with unlabeled TCP-1 peptide prior to the cell incubation with Cy7-PEG4-TCP-1, cell fluorescent signals were significantly reduced relative to the cells without blockade. Relative to Cy7-TCP-1, superior brilliance and visibility of fluorescence was observed in the tumor with Cy7-PEG4-TCP-1 and maintained up to 18h post-injection. [99mTc]Tc-HYNIC-PEG4-TCP-1 images in xenograft and orthotopic CRC models demonstrated that TCP-1 PEGylation preserved tumor-targeting capability of TCP-1, but its distribution (%ID/g) in the liver and intestine was higher than that of [99mTc]Tc-HYNIC-TCP-1 (1.51 ± 0.29 vs 0.53 ± 0.12, P < 0.01). Better tumor visualization by [99mTc]Tc-HYNIC-TCP-1 was observed in the orthotopic CRC model due to lower intestinal radioactivity. TCP-1-based probes undergo endocytosis and localize in the cytoplasm and nucleus of human and mouse CRC cells. Tumor detectability of fluorescent TCP-1 peptide with a PEG4 spacer is promising due to its enhanced tumor binding affinity and rapid clearance kinetics from nontumor tissues. Non-PEGylated [99mTc]Tc-HYNIC-TCP-1 exhibits lower nonspecific accumulation in the liver and gastrointestinal tract and might have better capability for detecting CRC lesions in clinical sites. TCP-1 may represent an innovative targeting molecule for detecting CRC noninvasively.

Enhancement of Anti-Tumoral Immunity by β-Casomorphin-7 Inhibits Cancer Development and Metastasis of Colorectal Cancer

β-Casomorphin-7 (BCM) is a degradation product of β-casein, a milk component, and has been suggested to affect the immune system. However, its effect on mucosal immunity, especially anti-tumor immunity, in cancer-bearing individuals is not clear. We investigated the effects of BCM on lymphocytes using an in vitro system comprising mouse splenocytes, a mouse colorectal carcinogenesis model, and a mouse orthotopic colorectal cancer model. Treatment of mouse splenocytes with BCM in vitro reduced numbers of cluster of differentiation (CD) 20+ B cells, CD4+ T cells, and regulatory T cells (Tregs), and increased CD8+ T cells. Administration of BCM and the CD10 inhibitor thiorphan (TOP) to mice resulted in similar alterations in the lymphocyte subsets in the spleen and intestinal mucosa. BCM was degraded in a concentration- and time-dependent manner by the neutral endopeptidase CD10, and the formed BCM degradation product did not affect the lymphocyte counts. Furthermore, degradation was completely suppressed by TOP. In the azoxymethane mouse colorectal carcinogenesis model, the incidence of aberrant crypt foci, adenoma, and adenocarcinoma was reduced by co-treatment with BCM and TOP. Furthermore, when CT26 mouse colon cancer cells were inoculated into the cecum of syngeneic BALB/c mice and concurrently treated with BCM and TOP, infiltration of CD8+ T cells was promoted, and tumor growth and liver metastasis were suppressed. These results suggest that by suppressing the BCM degradation system, the anti-tumor effect of BCM is enhanced and it can suppress the development and progression of colorectal cancer.

ZNF37A promotes tumor metastasis through transcriptional control of THSD4/TGF-β axis in colorectal cancer.

Poorly differentiated colorectal cancer (CRC) is characterized by aggressive invasion and stromal fibroblast activation, which results in rapid progression and poor therapeutic consequences. However, the regulatory mechanism involved remains unclear. Here, we showed that ZNF37A, a member of KRAB-ZFP family, was upregulated in poorly differentiated CRCs and associated with tumor metastasis. ZNF37A enhanced the metastatic potential of multiple CRC cell lines and promoted distant metastasis in an orthotopic CRC model. Further investigation attributed the ZNF37A-exacerbated metastasis to increased extracellular TGF-β and the consequent activation of cancer-associated fibroblasts (CAFs) in tumor microenvironment (TME). Mechanistically, ZNF37A formed a complex with KAP1 and bound to the promoter of THSD4, a TME modulator, to suppress its transcription, which is required for ZNF37A-mediated TGF-β activation and CRC metastasis. Collectively, our study indicates that ZNF37A promotes TGF-β signaling in CRC cells and activates CAFs by transcriptionally repressing THSD4 to drive CRC metastasis, implicating ZNF37A as a potential biomarker for CRC differentiation and progression.

Bufarenogin induces intrinsic apoptosis via Bax and ANT cooperation.

Toads have high medicinal value and have been used for medicinal purposes since the Tang Dynasty period (7th‐10th Century AD). Bufarenogin, an active anti‐tumor constituent of toad venom, shows anti‐tumor activity. In this study, we investigated the inhibitory effects of bufarenogin on the growth and metastasis of colorectal cancer (CRC), particularly its effects on mediating intrinsic signaling pathways that initiate apoptosis. An orthotopic CRC model was established in nude mice via surgical orthotopic implantation to investigate tumor growth. Immunohistochemistry, immunofluorescence, and Western blotting assays were performed to evaluate protein expression. The in vitro results revealed the anti‐proliferative effect of bufarenogin against CRC cells. Bufarenogin caused cell death via apoptosis, as revealed by Annexin V/7‐amino‐actinomycin D double staining, which was verified using a pan‐caspase inhibitor. Bufarenogin induced B‐cell lymphoma 2‐associated X protein (Bax)‐dependent intrinsic apoptosis, as demonstrated by mitochondrial translocation of Bax and cytoplasm release of HCT116 wild‐type cells and cytochrome C (soluble pro‐apoptotic factors). Additionally, we showed that adenine‐nucleotide translocator interacted with Bax. Bufarenogin induced intrinsic apoptosis through the cooperation of Bax and adenine‐nucleotide translocator and inhibited the metastasis and growth of orthotopical CRC cells.

Oral Metformin and Polymetformin Reprogram Immunosuppressive Microenvironment and Boost Immune Checkpoint Inhibitor Therapy in Colorectal Cancer

AbstractImmune checkpoint inhibitors (ICI), especially PD‐1/PD‐L1 inhibitors, are emerging as promising therapeutic options for patients with advanced tumors, but the response rate in the majority of colorectal cancer (CRC) patients remains low. Herein, the use of metformin (Met) and its polymeric form (Polymet) to enhance the response rate of immune checkpoint blockade therapy is reported. Oral Met and Polymet significantly enhance the antitumor effect of ICI therapy in a murine orthotopic CRC model. Further analyses reveal that Met and Polymet reprogram the immunosuppressive tumor microenvironment via activation of adenosine 5′‐monophosphate‐activated protein kinase and inhibition of the mammalian target of rapamycin signaling pathway, stimulating T cell infiltration into the tumor. Additionally, oral Met and Polymet change the abundance of key microbes in the intestinal environment, including increasing the Lactobacillus population, which should exert tumor‐suppressing activities in the CRC. These findings highlight the potential of combining Met or Polymet with cancer immunotherapy and provide a strategy for sensitizing CRC patients to immune checkpoint blockade therapy.

Bioinorganic hybrid bacteriophage for modulation of intestinal microbiota to remodel tumor-immune microenvironment against colorectal cancer.

Mounting evidence suggests that the gut microbiota contribute to colorectal cancer (CRC) tumorigenesis, in which the symbiotic Fusobacterium nucleatum (Fn) selectively increases immunosuppressive myeloid-derived suppressor cells (MDSCs) to hamper the host's anticancer immune response. Here, a specifically Fn-binding M13 phage was screened by phage display technology. Then, silver nanoparticles (AgNP) were assembled electrostatically on its surface capsid protein (M13@Ag) to achieve specific clearance of Fn and remodel the tumor-immune microenvironment. Both in vitro and in vivo studies showed that of M13@Ag treatment could scavenge Fn in gut and lead to reduction in MDSC amplification in the tumor site. In addition, antigen-presenting cells (APCs) were activated by M13 phages to further awaken the host immune system for CRC suppression. M13@Ag combined with immune checkpoint inhibitors (α-PD1) or chemotherapeutics (FOLFIRI) significantly prolonged overall mouse survival in the orthotopic CRC model.

Cancer Therapy: Integrated Combination Treatment Using a “Smart” Chemotherapy and MicroRNA Delivery System Improves Outcomes in an Orthotopic Colorectal Cancer Model (Adv. Funct. Mater. 28/2018)

Cancer Therapy: Integrated Combination Treatment Using a “Smart” Chemotherapy and MicroRNA Delivery System Improves Outcomes in an Orthotopic Colorectal Cancer Model (Adv. Funct. Mater. 28/2018)

Establishment of orthotopic colorectal cancer model by colonoscopy with submucosal injection in living mice

Objective To set up a living mice colonoscopy platform to establish an orthotopic model of colorectal cancer in mice under direct vision, and to observe its biological behavior such as metastasis. Methods Eighteen-week-old male C57/BL mice were anesthetized, and the intestinal lumen of the mice was examined by a self-developed living mice colonoscopy and Olympus URF-P5 ureteroscopy, respectively. The imaging effects of the two methods were compared. Human colon cancer HT-29 cells were injected into the colonic mucosa of BALB/c-nu mice under direct vision. The colonoscopy was performed on the 3rd, 7th and 15th day after the injection to observe the tumor formation in the intestinal lumen. The mice were sacrificed when the body weight decreased significantly or cachexia appeared, and then the abdominal cavity was examined including the tumor formation and metastasis. Results The self-developed living mice colonoscopy platform can provide clear vision of enteric cavity, and no mice died in the colonoscopy examination. In vivo subcutaneous injection of HT-29 cells in mice was performed with a perforation rate of 15%, a mortality rate of 33.3%, a tumor formation rate of 62.5%, an abdominal metastasis rate of 60%, a liver metastasis rate of 25%, and an abdominal wall transfer rate of 25%. Conclusion The self-developed mice colonoscopy platform can be used for the study of colorectum in living mice. The imaging effect is no less than that of Olympus URF-P5 ureteroscopy. In addition, an orthotopic colorectal cancer model can be established by this platform combing with submucosal injection technology. Key words: Living mice colonoscopy; Submucosal injection; T stage; Tumor microenvironment; Orthotopic colorectal cancer model

Integrated Combination Treatment Using a “Smart” Chemotherapy and MicroRNA Delivery System Improves Outcomes in an Orthotopic Colorectal Cancer Model

AbstractMesoporous silica nanoparticles (MSN) can load and deliver potentially synergistic anticancer agents such as small molecule cytotoxics (like doxorubicin, DOX) and nucleic acids (like microRNA, miRNA). However, these cargos have different underlying chemical properties so overcoming respective intracellular delivery barriers is a key consideration. Strategies to deliver DOX from MSN frequently employ pH‐driven mechanisms that are restricted to the acidic environment of lysosomes. Conversely, strategies to deliver miRNA make use of approaches that deliberately compromise lysosomal membrane integrity to enable cytosolic delivery of the payload. To reconcile these two needs (lysosomal delivery of DOX and intracellular delivery of miRNA), a new methodology by “weaving” polyethylenimine on the MSN surface through disulfide bonds to achieve superior delivery of chemotherapy (DOX) and miRNA therapy (using miRNA‐145) is developed. Furthermore, an active targeting strategy based on a peptide ligand with affinity to glucose‐regulated protein 78 (GRP78), a cell surface protein overexpressed in colorectal carcinoma, is developed. The active targeting approach results in enhanced synergistic antitumor effect both in vitro and in vivo in an orthotopic murine model of colorectal cancer. Taken together, this work demonstrates the capability and advantages of “smart” MSN delivery systems to deliver anticancer cargo appropriately to targeted cancer cells.

Targeted low-dose TNFα delivered by TCP-1 peptide exerts differential synergistic effects on anti-cancer actions of chemotherapeutic drugs

Minute amounts of tumor vasculature-targeted TNFα was reported to increase endothelial permeability, thereby accelerating drug penetration and increasing the therapeutic index of chemotherapeutic agents. This study aimed at assessing the synergistic anti-cancer effects of three chemotherapeutic drugs and tumor vasculature-targeted TNFα in a murine orthotopic colorectal cancer model. TNFα was firstly coupled with a vascular-homing peptide TCP-1 to form a TNFα derivative (TCP-1/TNFα). After pretreated with 1 ng TCP-1/TNFα, the three chemotherapeutic drugs exerted differential synergistic effects on anti-cancer actions and presented different intratumoral accumulation statuses. To further assess transvascular transport efficiency of these drugs, an in vitro system was carried out to mimic the endothelial cell monolayer. Results showed that 5-fluorouracil which exerted the best anti-cancer action and intratumoral accumulation status with preadministration of 1 ng TCP-1/TNFα had the highest transvascular transportation efficiency. This work would provide important information for designing clinical studies with vasculature-targeted TNFα in combination with chemotherapeutic drugs for optimal synergism.

A novel vascular-targeting peptide for gastric cancer delivers low-dose TNFα to normalize the blood vessels and improve the anti-cancer efficiency of 5-fluorouracil

Various vascular-targeted agents fused with tumor necrosis factor α (TNFα) have been shown to improve drug absorption into tumor tissues and enhance tumor vascular function. TCP-1 is a peptide selected through in vivo phage library biopanning against a mouse orthotopic colorectal cancer model and is a promising agent for drug delivery. This study further investigated the targeting ability of TCP-1 phage and peptide to blood vessels in an orthotopic gastric cancer model in mice and assessed the synergistic anti-cancer effect of 5-fluorouracil (5-FU) with subnanogram TNFα targeted delivered by TCP-1 peptide. In vivo phage targeting assay and in vivo colocalization analysis were carried out to test the targeting ability of TCP-1 phage/peptide. A targeted therapy for improvement of the therapeutic efficacy of 5-FU and vascular function was performed through administration of TCP-1/TNFα fusion protein in this model. TCP-1 phage exhibited strong homing ability to the orthotopic gastric cancer after phage injection. Immunohistochemical staining suggested that and TCP-1 phage/TCP-1 peptide could colocalize with tumor vascular endothelial cells. TCP-1/TNFα combined with 5-FU was found to synergistically inhibit tumor growth, induce apoptosis and reduce cell proliferation without evident toxicity. Simultaneously, subnanogram TCP-1/TNFα treatment normalized tumor blood vessels. Targeted delivery of low-dose TNFα by TCP-1 peptide can potentially modulate the vascular function of gastric cancer and increase the drug delivery of chemotherapeutic drugs.

Tumor location impacts immune response in mouse models of colon cancer.

Existing preclinical models of human colorectal cancer (CRC) that rely on syngeneic subcutaneous grafts are problematic, because of increasing evidence that the immune microenvironment in subcutaneous tissue is significantly different from the gastrointestinal tract. Similarly, existing orthotopic models that use a laparotomy for establishing grafts are also problematic, because the surgical procedure results in extensive inflammation, thereby creating a nonphysiologic tumor microenvironment. To facilitate the bench-to-bedside translation of CRC immunotherapy strategies, we developed a novel orthotopic model in mice that uses endoscopy-guided microinjection of syngeneic cancer cells. When we compared immune system infiltration, we found that tumors in the subcutaneous model had fewer T cells, B cells, and natural killer (NK) cells, but more immunosuppressive myeloid cells; in contrast, tumors in our orthotopic model had a higher number of tumor-infiltrating T cells, B cells, and NK cells, with fewer immunosuppressive myeloid cells. The number of immune-stimulating cytokines, such as interleukin (IL)-2, IL-6, interferon (IFN)-gamma, and granzyme B, was also higher in tumors in our model, as compared with the subcutaneous model. Those differences resulted in heightened sensitivity to immune checkpoint blockade therapy in our endoscopy-guided orthotopic CRC model. Our study indicates that tumor location affects immune response in CRC mouse models; choosing the appropriate preclinical model is important when testing immunotherapy in CRC.

Near-infrared light triggered drug delivery system for higher efficacy of combined chemo-photothermal treatment.

In case of chemo-photothermal combination therapy, the synchronism of treatments plays an important role in achieving expected antitumor efficiency. In this study, a light triggered ROS mediated drug delivery system was developed with the help of ROS-sensitive moieties of arylboronic ester and ROS producer of ICG. We innovatively make use of the ROS production capability of ICG under NIR laser irradiation to promote a faster release of DOX resulting from swelling of PCH-DI due to the presence of arylboronic ester. Intracellular ROS detection demonstrated that ROS level of PCH-I increased under irradiation. Moreover, the faster release behavior of DOX from PCH-DI with NIR laser irradiation was confirmed by the in vitro drug release and cellular uptake study. Meanwhile, local hyperthermia was verified by photothermal effect tests. Therefore, the synchronism of the combination therapy was achieved via light triggered faster release of DOX (chemo-therapy) and local hyperthermia (thermal-therapy) using PCH-DI under irradiation. It was reasonable to attribute the efficient anti-tumor efficiency of PCH-DI both in vitro and in vivo to the enhanced synergistic effect of chemo-photothermal combination therapy with realization of synchronism. To this end, this novel co-delivery system has provided a promising solution for achieving the synchronism of treatment to strengthen the efficiency of combination therapy.

Leukocyte populations and IL-6 in the tumor microenvironment of an orthotopic colorectal cancer model.

Colorectal cancer (CRC) is a major health problem worldwide. It is often diagnosed late due to its asymptomatic nature. As with all cancers, an immune reaction is involved; however, in CRC, it is unknown if this immune response is favorable or unfavorable for disease progression. In this study, the immune response in mesenteric lymph nodes (MLNs) and Peyer's patches was investigated during development of CRC in an orthotopic mouse model. CRC was induced by injecting CT26 cells into the cecum wall of BALB/c mice. Flow cytometry was used to analyze leukocyte populations involved in tumor immunity in MLNs and Peyer's patches. Cryostat sections for immunohistochemistry were prepared from the caecum and colon from CRC-induced and sham-operated animals. Cytokines produced by mouse CT26 cell line were measuredin vitroandin vivo Significant increases in the number of CD8(+)/TCR(+)and CD49b(+)/TCR(-)(natural killer) cells were found in MLNs and Peyer's patches in the CRC group. In addition, γδT cells were present in the lamina propria of the colon tissues from sham-operated mice, but absent in the colon tissues from mice with CRC. Immunohistochemical analysis of tumorous tissues showed eosinophil, CD69(+)T cell, and CD11b(+)cell infiltration. Bothin vitroandin vivoCT26 tumor cells were interleukin (IL)-6 positive. In addition, tumor-infiltrating CD45(+)cells were also IL-6 positive. In summary, the kinetics of the immune response to CRC and the key effector lymphocytes that are implicated in tumor immunity are demonstrated. Furthermore, IL-6 is a key cytokine present within the tumor microenvironment.