Tumor-infiltrating monocytic myeloid-derived suppressor cells contribute to the development of an immunosuppressive tumor microenvironment in gastric cancer.

BACKGROUND: Gastric cancer (GC) is characterized by an immunosuppressive and treatment-resistant tumor immune microenvironment (TIME). Here, we investigated the roles of different immunosuppressive cell types in the development of the GC TIME. METHODS: Single-cell RNA sequencing (scRNA-seq) and multiplex immunostaining of samples from untreated or immune checkpoint inhibitor (ICI)-resistant GC patients were used to examine the correlation between certain immunosuppressive cells and the prognosis of GC patients. RESULTS: The results of the scRNA-seq analysis revealed that tumor-infiltrating monocytic myeloid-derived suppressor cells (TI-M-MDSCs) expressed higher levels of genes with immunosuppressive functions than other immunosuppressive cell types. Additionally, M-MDSCs in GC tissues expressed significantly higher levels of these markers than adjacent normal tissues. The M-MDSCs were most enriched in GC tissues relative to adjacent normal tissues. Among the immunosuppressive cell types assessed, the M-MDSCs were most enriched in GC tissues relative to adjacent normal tissues; moreover, their presence was most strongly associated with a poor prognosis. Immediate early response 3 (IER3), which we identified as a differentially expressed gene between M-MDSCs of GC and adjacent normal tissues, was an independent poor prognostic factor in GC patients (P = 0.0003). IER3+ M-MDSCs expressed higher levels of genes with immunosuppressive functions than IER3- M-MDSCs and were abundant in treatment-resistant GC patients. CONCLUSIONS: The present study suggests that TI-M-MDSCs, especially IER3+ ones, may play a predominant role in the development of the immunosuppressive and ICI-resistant GC TIME.

Beckwith-Wiedemann syndrome with juvenile fibrous nodules and lobular breast tumors: a case report and review of the literature.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is a genomic imprinting disorder caused by diverse genetic and/or epigenetic disorders of chromosome 11p15.5. BWS presents with a variety of clinical features, including overgrowth and an increased risk of embryonal tumors. Notably however, reports of patients with BWS and breast tumors are rare, and the association between these conditions is still unclear. Insulin-like growth factor-2 (IGF2) expression is known to be associated with the development of various cancers, including breast cancer, and patients with BWS with specific subtypes of molecular defects are known to show characteristic clinical features and IGF2 overexpression. CASE PRESENTATION: A 17-year-old girl who had been diagnosed with BWS based on an umbilical hernia, hyperinsulinemia, and left hemihypertrophy at birth, visited our department with a gradually swelling left breast. Her left breast was markedly larger than her right breast on visual examination. Imaging examinations showed two tumors measuring about 10 cm each in the left breast, and she was diagnosed with juvenile fibroadenoma following core needle biopsy. The two breast tumors were removed surgically and the patient remained alive with no recurrence. The final diagnosis was juvenile fibroadenoma without malignant findings. Immunohistochemical staining using IGF2 antibody revealed overexpression of IGF2 in the cytoplasm of ductal epithelial cells. Because of her clinical features and IGF2 overexpression, molecular defects of 11p15.5 including a possible genetic background of paternal uniparental disomy of chromosome 11 or hypermethylation of imprinting center 1 was suspected. CONCLUSIONS: In this case, overexpression of IGF2 suggested a possible relationship between BWS and breast tumors. Moreover, the characteristic clinical features and IGF2 staining predicted the subtype of 11p15.5 molecular defects in this patient.

Microenvironmental changes in familial adenomatous polyposis during colorectal cancer carcinogenesis.

Familial adenomatous polyposis (FAP) is a heritable disease that increases the risk of colorectal cancer (CRC) development because of heterozygous mutations in APC. Little is known about the microenvironment of FAP. Here, single-cell RNA sequencing was performed on matched normal tissues, adenomas, and carcinomas from four patients with FAP. We analyzed the transcriptomes of 56,225 unsorted single cells, revealing the heterogeneity of each cell type, and compared gene expression among tissues. Then we compared the gene expression with that of sporadic CRC. Furthermore, we analyzed specimens of 26 FAP patients and 40 sporadic CRC patients by immunohistochemistry. Immunosuppressiveness of myeloid cells, fibroblasts, and regulatory T cells was upregulated even in the early stages of carcinogenesis. CD8+ T cells became exhausted only in carcinoma, although the cytotoxicity of CD8+ T cells was gradually increased according to the carcinogenic step. When compared with those in the sporadic CRC microenvironment, the composition and function of each cell type in the FAP-derived CRC microenvironment had differences. Our findings indicate that an immunosuppressive microenvironment is constructed from a precancerous stage in FAP.

Homologous Recombination Repair Gene Alterations Are Associated with Tumor Mutational Burden and Survival of Immunotherapy.

BACKGROUND: Comprehensive genomic profiling (CGP) has become generally accepted practice in cancer care since CGP has become reimbursed by national healthcare insurance in Japan in 2019. However, its usefulness for cancer patients is insufficient for several reasons. METHODS: In an observational clinical study of FoundationOne® CDx, potential biomarkers were explored and the cause of testing failure was investigated. A total of 220 cancer patients were enrolled in the study during the period from 2018 to 2019 at Kyushu University Hospital. RESULTS: The primary tumor sites of the 220 cases were breast (115), colon (29), stomach (19), and pancreas (20). The present dataset suggested that homologous recombination repair (HRR) gene alterations were positively associated with tumor mutational burden-high (TMB-high) (p = 0.0099). A public dataset confirmed that patients with HRR gene alterations had a higher TMB and showed significantly longer survival of immunotherapy. In the present study, 18 cases failed sequencing. A lower percentage of tumor cell nuclei was the most common reason for testing failures (p = 0.037). Cases that received neoadjuvant chemotherapy before sampling tended to fail testing. CONCLUSIONS: HRR gene alterations can be a potential biomarker predicting TMB-high and a good response to immunotherapy. For successful sequencing, samples with lower percentages of tumor cell nuclei and previous neoadjuvant chemotherapy should be avoided.

Bone marrow-derived macrophages converted into cancer-associated fibroblast-like cells promote pancreatic cancer progression.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a desmoplastic reaction caused by cancer-associated fibroblasts (CAFs), which provokes treatment resistance. CAFs are newly proposed to be heterogeneous populations with different functions within the PDAC microenvironment. The most direct sources of CAFs are resident tissue fibroblasts and mesenchymal stem cells, however, the origins and functions of CAF subtypes remain unclear. Here, we established allogeneic bone marrow (BM) transplantation models using spontaneous PDAC mice, and then investigated what subtype cells derived from BM modulate the tumor microenvironment and affect the behavior of pancreatic cancer cells (PCCs). BM-derived multilineage hematopoietic cells were engrafted in recipient pancreas, and accumulated at the invasive front and central lesion of PDAC. We identified BM macrophages-derived CAFs in tumors. BM-derived macrophages treated with PCC-conditioned media expressed CAF markers. BM-derived macrophages led the local invasion of PCCs in vitro and enhanced the tumor invasive growth in vivo. Our data suggest that BM-derived cells are recruited to the pancreas during carcinogenesis and that the specific subpopulation of BM-derived macrophages partially converted into CAF-like cells, acted as leading cells, and facilitated pancreatic cancer progression. The control of the conversion of BM-derived macrophages into CAF-like cells may be a novel therapeutic strategy to suppress tumor growth.

Necroptosis in pancreatic cancer promotes cancer cell migration and invasion by release of CXCL5.

BACKGROUND: Necroptosis is a form of programmed cell death that is accompanied by release of intracellular contents, and reportedly contributes to various diseases. Here, we investigate the significance of necroptosis in pancreatic cancer. METHODS: We used immunohistochemistry and western blot analysis to evaluate expression of the key mediators of necroptosis-receptor-interacting serine/threonine protein kinase 3 (RIP3) and mixed lineage kinase domain-like (MLKL)-in human pancreatic cancer. We also tested the effects of conditioned media (CM) from necroptotic cells on pancreatic cancer cells in Transwell migration and Matrigel invasion assays. Protein array analysis was used to investigate possible mediators derived from necroptotic cells. RESULTS: RIP3 and MLKL are highly expressed in human pancreatic cancer tissues compared with normal pancreas. MLKL expression was particularly intense at the tumor invasion front. CM derived from necroptotic cells promoted cancer cell migration and invasion, but not CM derived from apoptotic cells. C-X-C motif chemokine 5 (CXCL5) was upregulated in CM derived from necroptotic cells compared with CM derived from control or apoptotic cells. Moreover, expression of the receptor for CXCL5, C-X-C-motif chemokine receptor-2 (CXCR2), was upregulated in pancreatic cancer cells. Inhibition of CXCR2 suppressed cancer cell migratory and invasive behavior enhanced by necroptosis. CONCLUSION: These findings indicate that necroptosis at the pancreatic cancer invasion front can promote cancer cell migration and invasion via the CXCL5-CXCR2 axis.

Neutrophil extracellular traps promote liver micrometastasis in pancreatic ductal adenocarcinoma via the activation of cancer­associated fibroblasts.

Cancer­associated fibroblasts (CAFs) promote the progression of pancreatic ductal adenocarcinoma (PDAC) via tumor­stromal interactions. Neutrophil extracellular traps (NETs) are extracellular DNA meshworks released from neutrophils together with proteolytic enzymes against foreign pathogens. Emerging studies suggest their contribution to liver metastasis in several types of cancer. Herein, in order to investigate the role of NETs in liver metastasis in PDAC, the effects of NET inhibitors on spontaneous PDAC mouse models were evaluated. It was demonstrated that DNase I, a NET inhibitor, suppressed liver metastasis. For further investigation, further attention was paid to liver micrometastasis and an experimental liver metastasis mouse model was used that was generated by intrasplenic tumor injection. Furthermore, DNase I also suppressed liver micrometastasis and notably, CAFs accumulated in metastatic foci were significantly decreased in number. In vitro experiments revealed that pancreatic cancer cells induced NET formation and consequently NETs enhanced the migration of hepatic stellate cells, which was the possible origin of CAFs in liver metastasis. On the whole, these results suggest that NETs promote liver micrometastasis in PDAC via the activation of CAFs.