We prepared Arabidopsis thaliana lines expressing a functional green fluorescent protein (GFP)-linked vacuolar H + -pyrophosphatase (H + -PPase) under the control of its own promoter to investigate morphological dynamics of vacuoles and tissue-specific expression of H + -PPase. The lines obtained had spherical structures in vacuoles with strong fluorescence, which are referred to as bulbs. Quantitative analyses revealed that the occurrence of the bulbs correlated with the amount of GFP. Next, we prepared a construct of H + -PPase linked with a nondimerizing GFP (mGFP); we detected no bulbs. These results indicate that the membranes adhere face-to-face by antiparallel dimerization of GFP, resulting in the formation of bulbs. In plants expressing H + -PPase-mGFP, intravacuolar spherical structures with double membranes, which differed from bulbs in fluorescence intensity and intermembrane spacing, were still observed in peripheral endosperm, pistil epidermis and hypocotyls. Four-dimensional imaging revealed the dynamics of formation, transformation, and disappearance of intravacuolar spherical structures and transvacuolar strands in living cells. Visualization of H + -PPase-mGFP revealed intensive accumulation of the enzyme, not only in dividing and elongating cells but also in mesophyll, phloem, and nectary cells, which may have high sugar content. Dynamic morphological changes including transformation of vacuolar structures between transvacuolar strands, intravacuolar sheet-like structures, and intravacuolar spherical structures were also revealed.

Background-Intra-tumor heterogeneity implies that sub-populations of cancer cells that differ in genetic, phenotypic, or behavioral characteristics coexist in a single tumor 1,2. Tumor heterogeneity drives progression, metastasis and treatment resistance, but its relationship with tumor infiltrating immune cells is a matter of debate where some argue that tumors with high heterogeneity may generate neo-antigens that attract immune cells, and the others claim that immune cells provide selection pressure that shapes tumor heterogeneity 3,4. Here we sought to study the association between tumor heterogeneity and immune cells in a real-world cohort utilizing The Cancer Genome Atlas (TCGA). Methods-Mutant Allele Tumor Heterogeneity (MATH) was calculated to estimate intra-tumoral heterogeneity, and immune cell compositions were estimated by CIBERSORT. Survival analyses were demonstrated by Kaplan Meir curves. Results-Tumors with high heterogeneity (High MATH) associated with worse overall survival (p=0.049) as well as ER+ (p=0.011) and non-triple negative tumors (p=0.01). High MATH tumors associated with less infiltration of anti-tumor CD8 (p<0.013) and CD4 T cells (p<0.00024), more tumor promoting regulatory T cells (p<4e-04), lower expression of T cell exhaustion markers; PDL-1 (p=0.0031), IDO2 (p=0.34), ADORA2A (p=0.018), VISTA (p=0.00013), and CCR4 (p<0.00001), lower expression of cytolytic enzymes granzyme-A (p=0.0056) and perforin 1 (p=0.053) as well as low cytolytic activity score (p=0.0028). Conclusions-High heterogeneity tumors are associated with less immune cell infiltration, less activation of the immune response, and worse survival in breast cancer. Our results support the notion that tumor heterogeneity is shaped by selection pressure of tumor infiltrating immune cells.

CD8 T cell is an essential component of tumor-infiltrating lymphocytes (TIL) and tumor immune microenvironment (TIME). Using the xCell CD8 T cell score of whole tumor gene expression data, we estimated these cells in total of 3837 breast cancer patients from TCGA, METABRIC and various GEO cohorts. The CD8 score correlated strongly with expression of CD8 genes. The score was highest for triple-negative breast cancer (TNBC), and a high score was associated with high tumor immune cytolytic activity and better survival in TNBC but not other breast cancer subtypes. In TNBC, tumors with a high CD8 score had enriched expression of interferon (IFN)-α and IFN-γ response and allograft rejection gene sets, and greater infiltration of anti-cancerous immune cells. The score strongly correlated with CD4 memory T cells in TNBC, and tumors with both a high CD8 score and high CD4 memory T cell abundance had significantly better survival. Finally, a high CD8 score was significantly associated with high expression of multiple immune checkpoint molecules. In conclusion, a high CD8 T cell score is associated with better survival in TNBC, particularly when tumor CD4 memory T cells were elevated. Our findings also suggest a possible use of the score as a predictive biomarker for response to immune checkpoint therapy.

Tumor associated macrophages (TAMs) play a critical role in biology of various cancers, including breast cancer. In the current study, we defined “M1” macrophage and “M1”/“M2” ratio by transcriptomic signatures using xCell. We investigated the association between high level of “M1” macrophage or “M1”/“M2” ratio and the tumor immune microenvironment by analyzing the transcriptome of publicly available cohorts, TCGA and METABRIC. We found that “M1” high tumors were not associated with prolonged survival compared with “M1” low tumors, or with the response to neoadjuvant chemotherapy. “M1” high tumors were associated with clinically aggressive features and “M1” high tumors enriched the cell proliferation and cell cycle related gene sets in GSEA. At the same time, “M1” high tumors were associated with high immune activity and favorable tumor immune microenvironment, as well as high expression of immune check point molecules. Strikingly, all these results were mirrored in “M1”/“M2” ratio high tumors. In conclusion, transcriptomically defined “M1” or “M1”/“M2” high tumors were associated with aggressive cancer biology and favorable tumor immune microenvironment but not with survival benefit, which resembled only part of their conventional clinical characteristics.

While cancer cells gain aggressiveness by mutations, abundant mutations release neoantigens, attracting anti-cancer immune cells. We hypothesized that in breast cancer (BC), where mutation is less common, tumors with high mutation rates demonstrate aggressive phenotypes and attract immune cells simultaneously. High mutation rates were defined as the top 10% of the mutation rate, utilizing TCGA and METABRIC transcriptomic data. Mutation rate did not impact survival although high mutation BCs were associated with aggressive clinical features, such as more frequent in ER-negative tumors (p < 0.01), in triple-negative subtype (p = 0.03), and increased MKI-67 mRnA expression (p < 0.01) in both cohorts. Tumors with high mutation rates were associated with APOBEC3B and homologous recombination deficiency, increasing neoantigen loads (all p < 0.01). Cell proliferation and immune activity pathways were enriched in BCs with high mutation rates. Furthermore, there were higher lymphocytes and M1 macrophage infiltration in high mutation BCs. Additionally, T-cell receptor diversity, cytolytic activity score (CYT), and T-cell exhaustion marker expression were significantly elevated in BCs with high mutation rates (all p < 0.01), indicating strong immunogenicity. In conclusion, enhanced immunity due to neoantigens can be one of possible forces to counterbalance aggressiveness of a high mutation rate, resulting in similar survival rates to low mutation BCs. Accumulation of somatic mutations, or somatic genome instability, has been the principle of carcinogenesis; cancer cells stem from a clone that has gained the somatically acquired genetic abnormalities, leading to malignant transformation and further progression 1. With advances in next-generation sequencing technologies, clinical interest in assessing mutation burden and identifying specific mutations in certain cancer types has been growing to utilize targeted therapies or to assess tumor biology, such as FoundationOne genomic panel testing 2-4. Mutation rates are variable among cancer types and outliers with significantly high mutation burdens, hypermutation, do exist in many cancer types 5. Interestingly, the definition of hypermutation has been variable in the literature 5-7 , although the common definition is usually greater than 10-100 mutations per Mega base pairs (Mbps). Campbell and colleagues recently performed comprehensive analysis of hypermutation in various tumor types, providing more insight into tumor evolution and identifying possible clinically actionable mutation signatures 6. The etiology of hypermutation varies between cancer types; ultraviolet (UV) light is the significant cause of mutations in melanoma, while smoking causes the mutations in non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (SCC) 7,8. Whereas UV light and smoking are examples of exogenous mutagens, there are endogenous processes for mutation, such as microsatellite instability (MSI), and Apolipoprotein

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Neoadjuvant chemotherapy (NAC) had been developed as a systematic approach before definitive surgery for the treatment of locally advanced or inoperable breast cancer such as inflammatory breast cancer in the past. In addition to its impact on surgery, the neoadjuvant setting has a benefit of providing the opportunity to monitor the individual drug response. Currently, the subject of NAC has expanded to include patients with early-stage, operable breast cancer because it is revealed that the achievement of a pathologic complete response (pCR) is associated with excellent long-term outcomes, especially in patients with aggressive phenotype breast cancer. In addition, this approach provides the unique opportunity to escalate adjuvant therapy in those with residual disease after NAC. Neoadjuvant chemotherapy in breast cancer is a rapidly evolving topic with tremendous interest in ongoing clinical trials. Here, we review the improvements and further challenges in the NAC setting in translational breast cancer research.

APOBEC3 enzymes contribute significantly to DNA mutagenesis in cancer. These enzymes are also capable of converting C bases at specific positions of RNAs to U. However, the prevalence and significance of this C-to-U RNA editing in any cancer is currently unknown. We developed a bioinformatics workflow to determine RNA editing levels at known APOBEC3-mediated RNA editing sites using exome and mRNA sequencing data of 1040 breast cancer tumors. Although reliable editing determinations were limited due to sequencing depth, editing was observed in both tumor and adjacent normal tissues. For 440 sites (411 genes), editing was determinable for ≥5 tumors, with editing occurring in 0.6%–100% of tumors (mean 20%, SD 14%) at an average level of 0.6%–20% (mean 7%, SD 4%). Compared to tumors with low RNA editing, editing-high tumors had enriched expression of immune-related gene sets, and higher T cell and M1 macrophage infiltration, B and T cell receptor diversity, and immune cytolytic activity. Concordant with this, patients with increased RNA editing in tumors had better disease- and progression-free survivals (hazard ratio = 1.67–1.75, p < 0.05). Our study identifies that APOBEC3-mediated RNA editing occurs in breast cancer tumors and is positively associated with elevated immune activity and improved survival.