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Supplementary materials and methods

Figure S1. Schematic representation of workflow for single-cell isolation and sc-qPCR. Figure S2. Violin plot of each individual gene expression along drug resistance acquisition. Figure S3. Correlation of several genes expressed at the single-cell level, separated by cell type as represented by the color of each dot. Figure S4. Lef1, Vim and Cav1 protein expression in several Luminal breast cancer cell lines. Figure S5. Larger visual fields showing immunostaining in tissue samples. Figure S6. Clinical information regarding the role of Lef1 in stained tissue samples. Figure S7. Comparison of Lef1 protein levels in MCF7 and derivatives.

Table S1. List of TaqMan probes used for the 95-primer set.

Drug resistance is a major obstacle in the treatment of breast cancer. Surviving cells lead to tumor recurrence and metastasis, which remains the main cause of cancer-related mortality. Breast cancer is also highly heterogeneous, which hinders the identification of individual cells with the capacity to survive anticancer treatment. To address this, we performed extensive single-cell gene-expression profiling of the luminal-type breast cancer cell line MCF7 and its derivatives, including docetaxel-resistant cells. Upregulation of epithelial-to-mesenchymal transition and stemness-related genes and downregulation of cell-cycle–related genes, which were mainly regulated by LEF1, were observed in the drug-resistant cells. Interestingly, a small number of cells in the parental population exhibited a gene-expression profile similar to that of the drug-resistant cells, indicating that the untreated parental cells already contained a rare subpopulation of stem-like cells with an inherent predisposition toward docetaxel resistance. Our data suggest that during chemotherapy, this population may be positively selected, leading to treatment failure.Significance:This study highlights the role of breast cancer intratumor heterogeneity in drug resistance at a single-cell level.

A total of 100 patients were retrospectively analyzed with magnetic resonance imaging-ultrasonography (MRI-US) fusion biopsy(KOELIS, TRINITY®) at our institution between October 2019 and May 2020. The median patient age was 71 years, median prostate specific antigen (PSA) level was 7.4 ng/ml, and median PSA-density was 0.183 mg/ml. Sixty-one of the patients were positive for cancer ; 14 of them were positive by targeted biopsy only, 9 were positive by systematic biopsy only, and 38 were positive by both. Clinically significant prostate cancer (CPSC ; Gleason Score ≥3＋4 and % core ≥50%) was detected by target biopsies in 46 patients and by systematic biopsies in 33 patients. The positive core detection rate for CSPC was 32.5% for targeted biopsies and 7.0% for systematic biopsies(P＜0.0001), with a significantly higher rate for targeted biopsies. These results indicate that in MRI-US fusion biopsy, targeted biopsy has a higher detection rate for cancer and a significantly higher detection rate for clinically significant prostate cancer compared with systematic biopsy.

Background & Aims: There is a long-standing debate regarding the biological significance of polyploidy in hepatocytes. Recent studies have provided increasing evidence that hepatocytes with different ploidy statuses behave differently in a context-dependent manner (eg, susceptibility to oncogenesis, regenerative ability after injury, and in vitro proliferative capacity). However, their overall transcriptomic differences in a physiological context is not known. Methods: By using microarray transcriptome analysis, we investigated the heterogeneity of hepatocyte populations with different ploidy statuses. Moreover, by using single-cell quantitative reverse-transcription polymerase chain reaction (scPCR) analysis, we investigated the intrapopulational transcriptome heterogeneity of 2c and 4c hepatocytes. Results: Microarray analysis showed that cell cycle–related genes were enriched in 8c hepatocytes, which is in line with the established notion that polyploidy is formed via cell division failure. Surprisingly, in contrast to the general consensus that 2c hepatocytes reside in the periportal region, in our bulk transcriptome and scPCR analyses, the 2c hepatocytes consistently showed pericentral hepatocyte-enriched characteristics. In addition, scPCR analysis identified a subpopulation within the 2c hepatocytes that co-express the liver progenitor cell markers Axin2, Prom1, and Lgr5, implying the potential biological relevance of this subpopulation. Conclusions: This study provides new insights into hepatocyte heterogeneity, namely 2c hepatocytes are preferentially localized to the pericentral region, and a subpopulation of 2c hepatocytes show liver progenitor cell–like features in terms of liver progenitor cell marker expression (Axin2, Prom1, and Lgr5). Keywords: Hepatocyte, Ploidy, Zonation, Single-Cell PCR, Transcriptome

Liquid biopsy is indispensable for the resolution of current medical issues, such as the cost of developing new drugs and predicting responses of patients to drugs. In this sense, not only the technology for liquid biopsy but also the target biomolecules for biomarkers need to be identified. Extracellular vesicles (EVs), which contain various proteins, including membrane-bound proteins, and RNAs, including mRNA and long/short noncoding RNAs, have emerged as ideal targets for liquid biopsy. These complex biomolecules are covered by a lipid bilayer, which can protect them from degradation. In this review, we review current topics regarding EVs as cancer biomarkers and introduce technologies used for these recently emerged biomolecules., Cancer diagnosis: good things come in small packages Extracellular vesicles (EVs), small packets of proteins and RNAs secreted by cells and carried in the bloodstream, are promising diagnostic markers for cancer. Developing new markers that can be collected non-invasively can dramatically improve detection and treatment of cancer. EVs secreted by cells, including tumor cells, send messages to distant cells. EVs collected from blood samples can potentially be excellent disease markers. Takahiro Ochiya and Nobuyoshi Kosaka at Tokyo Medical University in Japan and co-workers have reviewed available EV technology. They report that tumors reliably secrete diagnostic EVs, revealing the origin and state of the tumor. Some EVs can even be used to predict whether a cancer might spread to another tissue. With improvement of methods for isolation and standardization, EVs could provide sensitive markers for early diagnosis of many cancers.

Bladder cancer is the 9th leading cause of cancer death worldwide. The major problem in bladder cancer is primarily the high recurrence rate after drug treatment and resection. Although conventional screening methods, such as cystoscopy, urinary cytology and ultrasound sonography, have become widely used in clinical settings, the diagnostic performance of these modalities is unsatisfactory due to low accuracy or high invasiveness. Because circulating micro RNA (miRNA) profiles have recently been reported as an attractive tool for liquid biopsy in cancer screening, here, we performed global miRNA profiling of 392 serum samples of bladder cancer patients with 100 non-cancer samples and 480 samples of other types of cancer as controls. We randomly classified the bladder cancer and control samples into 2 cohorts, a training set (N = 486) and a validation set (N = 486). By comparing both controls, we identified specific miRNA, such as miR-6087, for diagnosing bladder cancer in the training and validation sets. Furthermore, we found that a combination of 7 miRNA (7-miRNA panel: miR-6087, miR-6724-5p, miR-3960, miR-1343-5p, miR-1185-1-3p, miR-6831-5p and miR-4695-5p) could discriminate bladder cancer from non-cancer and other types of tumors with the highest accuracy (AUC: .97; sensitivity: 95%; specificity: 87%). The diagnostic accuracy was high, regardless of the stage and grade of bladder cancer. Our data demonstrated that the 7-miRNA panel could be a biomarker for the specific and early detection of bladder cancer.

A subset of prostate cancer displays a poor clinical outcome. Therefore, identifying this poor prognostic subset within clinically aggressive groups (defined as a Gleason score (GS) ≧8) and developing effective treatments are essential if we are to improve prostate cancer survival. Here, we performed a bioinformatics analysis of a TCGA dataset (GS ≧8) to identify pathways upregulated in a prostate cancer cohort with short survival. When conducting bioinformatics analyses, the definition of factors such as “overexpression” and “shorter survival” is vital, as poor definition may lead to mis-estimations. To eliminate this possibility, we defined an expression cutoff value using an algorithm calculated by a Cox regression model, and the hazard ratio for each gene was set so as to identify genes whose expression levels were associated with shorter survival. Next, genes associated with shorter survival were entered into pathway analysis to identify pathways that were altered in a shorter survival cohort. We identified pathways involving upregulation of GRB2. Overexpression of GRB2 was linked to shorter survival in the TCGA dataset, a finding validated by histological examination of biopsy samples taken from the patients for diagnostic purposes. Thus, GRB2 is a novel biomarker that predicts shorter survival of patients with aggressive prostate cancer (GS ≧8).

Drug resistance is a major obstacle in the treatment of breast cancer. Surviving cells lead to tumor recurrence and metastasis, which remains the main cause of cancer-related mortality. Breast cancer is also highly heterogeneous, which hinders the identification of individual cells with the capacity to survive anticancer treatment. To address this, we performed extensive single-cell gene-expression profiling of the luminal-type breast cancer cell line MCF7 and its derivatives, including docetaxel-resistant cells. Upregulation of epithelial-to-mesenchymal transition and stemness-related genes and downregulation of cell-cycle–related genes, which were mainly regulated by LEF1, were observed in the drug-resistant cells. Interestingly, a small number of cells in the parental population exhibited a gene-expression profile similar to that of the drug-resistant cells, indicating that the untreated parental cells already contained a rare subpopulation of stem-like cells with an inherent predisposition toward docetaxel resistance. Our data suggest that during chemotherapy, this population may be positively selected, leading to treatment failure. Significance: This study highlights the role of breast cancer intratumor heterogeneity in drug resistance at a single-cell level.

Background Bladder cancer is the most prevalent malignancy involving the urinary system and exhibits a markedly high recurrence rate. Therefore, reliable and noninvasive diagnostic and surveillance methods are desperately needed. Patients and Methods Candidate microRNAs (miRNAs) were selected from the miRNAs that were differentially expressed in bladder cancer cell lines (T24 and RT4) compared to normal ureteral epithelial tissue using miRNA-microarray analysis. The candidate miRNAs were validated by quantitative reverse transcription polymerase chain reaction assay using voided urine samples. Results We identified 3 miRNAs (miR-301b, -563, and -146a-5p) that demonstrated > 2-fold higher expression levels in cancer cell lines than in the normal ureteral epithelial tissue. Of these, only miR-146a-5p was consistently and significantly higher in urine samples from the patients with bladder cancer than in those from the normal individuals ( P = .0014). The patients with high-grade tumors exhibited significantly higher urinary miR-146a-5p levels than those with low-grade tumors, and the patients with invasive tumors tended to show higher urinary miR-146a-5p levels than those with noninvasive tumors. Elevated urinary miR-146a-5p levels in patients with bladder cancer were decreased to the normal level after transurethral resection of the tumors ( P = .0214). Conclusion Our study suggested that urinary miR-146a-5p might be useful as a new noninvasive diagnostic marker, therapeutic target, or anticancer agent for bladder cancer, as well as for increasing our understanding of cancer biology.

Drug resistance is a major problem for breast cancer patients. Docetaxel is an anti-mitotic agent that serves as first line of treatment in metastatic breast cancer, however it is susceptible to cellular drug resistance. Drug-resistant cells are able to spread during treatment, leading to treatment failure and eventually metastasis, which remains the main cause for cancer-associated death. In previous studies, we used single-cell technologies and identified a set of genes that exhibit increased expression in drug-resistant cells, and they are mainly regulated by Lef1. Furthermore, upregulating Lef1 in parental cells caused them to become drug resistant. Therefore, we hypothesized that inhibiting Lef1 could resensitize cells to docetaxel. Here, we confirmed that Lef1 inhibition, especially on treatment with the small molecule quercetin, decreased the expression of Lef1 and resensitized cells to docetaxel. Our results demonstrate that Lef1 inhibition also downregulated ABCG2, Vim, and Cav1 expression and equally decreased Smad-dependent TGF-&beta, signaling pathway activation. Likewise, these two molecules worked in a synergetic manner, greatly reducing the viability of drug-resistant cells. Prior studies in phase I clinical trials have already shown that quercetin can be safely administered to patients. Therefore, the use of quercetin as an adjuvant treatment in addition to docetaxel for the treatment of breast cancer may be a promising therapeutic approach.

Background The levels of corneal donation are insufficient to meet the demand for corneal transplantation in Japan. To overcome this problem, we started to routinely mention the possibility of corneal donation to the families of patients who died in our hospital's Urology Department in February 2008. In this study, we evaluated the effectiveness of this approach. Methods We retrospectively reviewed the medical records of the patients who died in the Department of Urology, St. Marianna University School of Medicine Hospital, and analyzed the patients' characteristics and information about corneal donation. Results In total, 211 patients died in our department between February 2008 and March 2017, and 155 patients were medically suitable corneal donors. We mentioned the possibility of corneal donation to 129 (83.2%) families, and 29 (18.7%) families agreed. Three families subsequently withdrew their consent. Finally, 26 (16.8%) of the families that were approached about corneal donation by urologists agreed to donate their relatives' corneas. Another 2 families voluntarily offered to donate their relatives' corneas. Thus, 28 (18.1%) of 155 medically suitable donors donated their corneas for transplantation. Twenty-six (92.8%) donors were 60 years or older and all donors were affected with malignant genitourinary tumors. Fifty-four (96.4%) corneas were successfully transplanted into recipients. Conclusions Even elderly patients who die of solid carcinoma can be an important source of corneal donors. In this study, we showed that routine referral by urologists increased corneal donation. If this approach were adopted by other departments, it might further increase the number of corneal donations.

In recent years, the frequency of high-risk kidney transplantations has increased. We report a case in which a 72-year-old man with various severe comorbidities (prostate cancer, diabetes mellitus, complete atrioventricular block, coronary artery stenosis, severe stenosis of the popliteal arteries, and severe calcification of the iliac arteries) who received an orthotopic kidney transplantation. To prevent the occurrence of acute limb ischemia due to the steal phenomenon (caused by the kidney graft), we decided that a heterotopic kidney transplantation involving the iliac arteries was not an appropriate option. Therefore, as an alternative, left native nephrectomy was performed followed by an orthotopic kidney transplantation to the native renal artery and renal vein through a left subcostal incision. Postoperative ureteral stenosis occurred, and so stent exchange was required every 6 months. Despite the ureteral complication, the patient's serum creatinine level was 1.5 mg/dL at 2 years after the procedure.

Solitary fibrous tumor (SFT) of the kidney is a rare entity and usually displays a favorable prognosis. We herein report a second case of renal SFT developing local recurrence. A 50-year-old man was referred to our hospital because of a left renal mass. An abdominal CT detected a large renal tumor and radical nephrectomy was performed with a possible diagnosis of renal cell carcinoma. The resected tumor size was measured at 17 × 11 × 8 cm. Grossly, necrosis was observed in central lesion of the tumor but hemorrhage was not observed. Microscopically, the tumor consisted of spindle-shaped cells with scant cytoplasm accompanied by hyalinized collagenous tissue, which displayed hemangiopericytomatous patterns. The cellularity was normal and nuclear pleomorphism was not observed. Ki-67 labeling index was less than 3%. The pathological diagnosis of SFT was made without obvious malignant findings. Three years after the surgery, a follow-up CT scan detected a mass lesion in the tumor bed. Surgical resection was performed and the resected tumor was compatible with local recurrence of the SFT without obvious malignant findings. Renal SFT should be carefully monitored even in the absence of obvious malignant findings.

We report the first case in Japan of paraneoplastic dermatomyositis with pure seminoma, a tumor that extremely rarely accompanies dermatomyositis. The patient presented to the hospital with muscle weakness and erythema and was diagnosed with dermatomyositis from skin biopsy. Routine radiological screening revealed testicular tumor and massive lymph node metastases. We initially performed orchiectomy along with conventional immunotherapy. However, muscle weakness gradually worsened, and he eventually showed dysphagia and forced respiration and became bedridden. Although he seemed close to being too unstable to tolerate further treatment, we started carefully adjusted chemotherapy comprising 4 courses of etoposide plus cisplatin, which proved highly successful. Lymph node metastases completely disappeared and swallowing and respiration fully normalized after completing chemotherapy. We believe that this clinical success was due to our decision to initiate chemotherapy even in such a weak patient.