Analysis of anti-tumor effect and mechanism of GLS1 inhibitor CB-839 in colorectal cancer using a stroma-abundant tumor model.

BACKGROUND: Glutaminase 1 (GLS1), a key enzyme in glutamine metabolism in cancer cells, acts as a tumor promoter and could be a potential therapeutic target. CB-839, a GLS1-specific inhibitor, was developed recently. Herein, we aimed to elucidate the anti-tumor effects and mechanism of action of CB-839 in colorectal cancer (CRC). METHODS: Using the UCSC Xena public database, we evaluated GLS1 expression in various cancers. Immunostaining for GLS1 was performed on 154 surgically resected human CRC specimens. Subsequently, we examined the GLS1 mRNA expression levels in eight CRC cell lines and evaluated the association between GLS1 expression and CB-839 efficacy. To create a reproducible CRC model with abundant stroma and an allogeneic immune response, we co-transplanted CT26 and stem cells into BALB/c mice and treated them with CB-839. Finally, RNA sequencing of mouse tumors was performed. RESULTS: Database analysis showed higher GLS1 expression in CRC tissues than in normal colon tissues. Clinical samples from 114 of the 154 patients with CRC showed positive GLS1 expression. GLS1 expression in clinical CRC tissues correlated with vascular invasion. CB-839 treatment inhibited cancer cell proliferation depending on GLS1 expression in vitro and inhibited tumor growth and metastasis in the CRC mouse model. RNA sequencing revealed that CB-839 treatment inhibited stromal activation, tumor growth, migration, and angiogenesis. These findings were validated through in vitro and in vivo experiments and clinical specimen analysis. CONCLUSIONS: GLS1 expression in CRC plays important roles in tumor progression. CB-839 has inhibitory effects on cancer proliferation and the tumor microenvironment.

Pexidartinib and Immune Checkpoint Inhibitors Combine to Activate Tumor Immunity in a Murine Colorectal Cancer Model by Depleting M2 Macrophages Differentiated by Cancer-Associated Fibroblasts.

Tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs) are known to play supportive roles in tumor development and progression, but their interactions in colorectal cancer (CRC) remain unclear. Here, we investigated the effects of colon-cancer-derived CAFs on TAM differentiation, migration, and tumor immunity, both in vitro and in vivo. When co-cultured with monocytes, CAFs attracted monocytes and induced their differentiation into M2 macrophages. Immunohistology of surgically resected human CRC specimens and orthotopically transplanted mouse tumors revealed a correlation between numbers of CAFs and numbers of M2 macrophages. In a mouse model of CRC orthotopic transplantation, treatment with an inhibitor of the colony-stimulating factor-1 receptor (PLX3397) depleted M2 macrophages and increased CD8-positive T cells infiltrating the tumor nest. While this treatment had a minor effect on tumor growth, combining PLX3397 with anti-PD-1 antibody significantly reduced tumor growth. RNA-seq following combination therapy showed activation of tumor immunity. In summary, CAFs are involved in the induction and mobilization of M2 macrophage differentiation in the CRC tumor immune microenvironment, and the combination of cancer immunotherapy and PLX3397 may represent a novel therapeutic option for CRC.

Pazopanib-induced enteritis in a patient with renal cell carcinoma.

A 69-year-old woman presented to our department with the chief complaint of diarrhea. She had undergone left nephrectomy for renal cancer 14 years earlier. Three years earlier, metastasis was detected in the left retroperitoneal cavity, and pazopanib administration was initiated. In the 29th month after the start of chemotherapy, the patient developed diarrhea, and on the 31st month, computed tomography showed thickening of the intestinal wall. Colonoscopy revealed white villi, intramucosal hemorrhage in the terminal ileum, and rough inflammatory mucosa with inflammatory polyps extending from the transverse to the sigmoid colon. Suspecting pazopanib-induced enteritis, we discontinued the medication, and the diarrhea resolved within 3 days. On the 21st day after discontinuation, colonoscopy revealed that the inflammatory polyps had shrunk, and the inflammatory findings had improved. Biopsy of the white villi of the ileum revealed histiocytes. The patient resumed treatment with pazopanib at 400 mg/day and developed soft stool on the 7th day after resumption. Compared with other tyrosine-kinase inhibitor-induced enteritis cases, this case showed less bleeding and more extensive inflammatory findings. There are similarities as well as differences from cases of previously reported pazopanib-induced enteritis. The mechanisms and characteristics of this disease require further investigation.

WEE1 Inhibitor Adavosertib Exerts Antitumor Effects on Colorectal Cancer, Especially in Cases with p53 Mutations.

Inhibition of WEE1, a key regulator of the G2/M checkpoint of the cell cycle, induces apoptosis by initiating mitosis without repairing DNA damage. However, the effects of WEE1 inhibitors on the tumor immune microenvironment in colorectal cancer (CRC) remain unclear. Here, we investigated the association between WEE1 expression and CRC clinicopathological features using surgically resected CRC specimens and assessed the antitumor effects of a WEE1 inhibitor using CRC cell lines and orthotopic transplantation mouse models. WEE1 expression was not correlated with the clinicopathological features of CRC. The WEE1 inhibitor suppressed cell proliferation in a concentration-dependent manner in all CRC cell lines. It also increased the percentage of cells in the G2/M phase and apoptotic cells, especially in cell lines with p53 mutations, but did not alter these cell percentages in most p53 wild-type cell lines. In the orthotopic mouse model of CRC, tumor volume was significantly reduced in the WEE1 inhibitor-treated group compared to that in the control group. RNA sequencing and immunohistochemistry analyses of mouse tumors revealed that treatment with the WEE1 inhibitor activated tumor immunity and suppressed stromal reactions. These results demonstrate the potential antitumor effects of WEE1 inhibitors in CRC, particularly in patients with p53 mutations.

The Anti-Tumor Effect of the Newly Developed LAT1 Inhibitor JPH203 in Colorectal Carcinoma, According to a Comprehensive Analysis.

A novel large neutral amino acid transporter 1 (LAT1)-specific inhibitor, JPH203, is expected to cause cancer-specific starvation and possess anti-tumor effects; however, its anti-tumor mechanism for colorectal cancer (CRC) remains unclear. We analyzed LAT family gene expressions in public databases using UCSC Xena and evaluated LAT1 protein expression using immunohistochemistry in 154 cases of surgically resected CRC. We also evaluated mRNA expression using polymerase chain reaction in 10 CRC cell lines. Furthermore, JPH203 treatment experiments were conducted in vitro and in vivo using an allogeneic immune-responsive mouse model with abundant stroma created via the orthotopic transplantation of the mouse-derived CRC cell line CT26 and mesenchymal stem cells. The treatment experiments were followed by comprehensive gene expression analyses with RNA sequencing. Database analyses and immunohistochemistry research on clinical specimens revealed that LAT1 expression was cancer-dominant, and its increase was accompanied by tumor progression. In vitro, JPH203 was effective in an LAT1 expression-dependent manner. In vivo, JPH203 treatment considerably reduced tumor size and metastasis, and RNA sequencing-based pathway analysis showed that not only tumor growth and amino acid metabolism pathways, but also stromal activation-related pathways were suppressed. The results of the RNA sequencing were validated in the clinical specimens, as well as both in vitro and in vivo. LAT1 expression in CRC plays an important role in tumor progression. JPH203 may inhibit the progression of CRC and tumor stromal activity.

Anti-tumor necrosis factor-alpha monoclonal antibody suppresses colorectal cancer growth in an orthotopic transplant mouse model.

The risk of malignant tumor progression has been a concern associated with the use of anti-tumor necrosis factor-alpha monoclonal antibody (anti-TNFα mAb). On the contrary, recent observational studies have reported negatively on this risk and instead suggested that anti-TNFα mAb acts as a tumor suppressor in inflammatory carcinogenesis models and subcutaneous transplant models of colorectal cancer. However, no consensus has been established regarding the actual effects of anti-TNFα mAb on malignant tumors. Here, we aimed to evaluate, for the first time, the effect of anti-TNFα mAb on the tumor microenvironment in the absence of intestinal inflammation in a colorectal cancer orthotopic transplant mouse model suitable for tumor microenvironment assessment. The orthotopic transplantation model was developed by transplanting CT26 cells into the cecum of BALB/c mice. Changes in tumor size and weight were recorded 3 weeks after transplantation, and the tumor microenvironment was assessed via RNA sequencing and immunohistological staining. In the orthotopic transplant model, the administration of anti-TNFα mAb led to a reduction in colorectal cancer. The RNA sequencing analysis showed upregulation of immune-related pathways and apoptosis and suppression of stromal- and tumor growth-related pathways. Additionally, Gene Ontology analysis showed inhibition of angiogenesis. Immunohistochemical staining showed inhibition of tumor growth, increase in apoptosis, suppression of stromal response, suppression of angiogenesis, enhancement of tumor immunity, and reduction in the number of tumor-associated macrophages. Anti-TNFα mAb acts as an inhibitor of tumor progression in the tumor microenvironment of a colorectal cancer orthotopic transplant mouse model.