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Osteosarcoma (OS) is a highly malignant bone tumor and the prognosis for non‐responders to chemotherapy remains poor. Previous studies have shown that human sarcomas contain sarcoma‐initiating cells (SIC), which have the characteristics of high tumorigenesis and resistance to chemotherapy. In the present study, we characterized SIC of a novel OS cell line, screened for SIC‐related genes, and tried to regulate the proliferation of OS by metabolic interference. Initially, we established a new human OS cell line (OS13) and isolated clones showing higher tumorigenesis as SIC (OSHIGH) and counterpart clones. OSHIGH cells showed chemoresistance and their metabolism highly depended on aerobic glycolysis and suppressed oxidative phosphorylation. Using RNA‐sequencing, we identified LIN28B as a SIC‐related gene highly expressed in OSHIGH cells. mRNA of LIN28B was expressed in sarcoma cell lines including OS13, but its expression was not detectable in normal organs other than the testis and placenta. LIN28B protein was also detected in various sarcoma tissues. Knockdown of LIN28B in OS13 cells reduced tumorigenesis, decreased chemoresistance, and reversed oxidative phosphorylation function. Combination therapy consisting of a glycolysis inhibitor and low‐dose chemotherapy had antitumor effects. In conclusion, manipulation of glycolysis combined with chemotherapy might be a good adjuvant treatment for OS. Development of immunotherapy targeting LIN28B, a so‐called cancer/testis antigen, might be a good approach. Osteosarcoma‐initiating cell antigen LIN28B was expressed in sarcoma tissues. ES, epithelioid sarcoma; MFS, myxofibrosarcoma; OS, osteosarcoma; SS, synovial sarcoma.

Photodynamic therapy (PDT) using the photosensitizer talaporfin sodium (talaporfin) is a new mode of treatment for cancer. However, the metabolic mechanism of talaporfin has not been clarified. Thus, we investigated the uptake, transportation, and elimination mechanisms of talaporfin in carcinoma and sarcoma. The results showed that talaporfin co‐localized in early endosomes and lysosomes. Talaporfin uptake was via clathrin‐ and caveolae‐dependent endocytosis and a high amount of intracellular ATP was essential. Inhibition of lysosomal enzymes maintained intracellular talaporfin levels. Inhibition of K‐Ras signaling reduced talaporfin uptake in carcinoma and sarcoma cell lines. Talaporfin was taken up by clathrin‐ and caveolae‐dependent endocytosis, translocated from early endosomes to lysosomes, and finally degraded by lysosomes. We also demonstrated that ATP is essential for the uptake of talaporfin and that activation of K‐Ras is involved as a regulatory mechanism. These results provide new insights into the metabolism of talaporfin in cancer cells for the enhancement of PDT for carcinoma and sarcoma. Intracellular talaporfin was co‐localized with early endosomes and lysosomes but not with mitochondria in osteosarcoma cells.

Background Peptide-vaccination therapy targeting tumour-associated antigens can elicit immune responses, but cannot be used to eliminate large tumour burden. In this study, we developed a therapeutic single-chain variable-fragment (scFv) antibody that recognises the cancer stem-like cell/cancer-initiating cell (CSC/CIC) antigen, DNAJB8. Methods We screened scFv clones reacting with HLA-A24:20/DNAJB8-derived peptide (DNAJB8_143) complex using naive scFv phage-display libraries. Reactivity and affinity of scFv clones against the cognate antigen were quantified using FACS and surface plasmon resonance. Candidate scFv clones were engineered to human IgG1 (hIgG1) and T-cell-engaging bispecific antibody (CD3xJB8). Complement-dependent cytotoxicity (CDC) and bispecific antibody-dependent cellular cytotoxicity (BADCC) were assessed. Results scFv clones A10 and B10 were isolated after bio-panning. Both A10-hIgG1 and B10-hIgG1 reacted with DNAJB8-143 peptide-pulsed antigen-presenting cells and HLA-A24(+)/DNAJB8(+) renal cell carcinoma and osteosarcoma cell lines. A10-hIgG1 and B10-hIgG1 showed strong affinity with the cognate HLA/peptide complex ( K D  = 2.96 × 10 −9  M and 5.04 × 10 −9  M, respectively). A10-hIgG1 and B10-hIgG1 showed CDC against HLA-A24(+)/DNAJB8(+) cell lines. B10-(CD3xJB8) showed superior BADCC to A10-(CD3xJB8). Conclusion We isolated artificial scFv antibodies reactive to CSC/CIC antigen DNAJB8-derived peptide naturally present on renal cell carcinoma and sarcoma. Immunotherapy using these engineered antibodies could be promising.

We developed an immunohistogram representing an individual cancer-immunity cycle based on immunohistochemical analyses. We evaluated its ability to predict the efficacy of immune checkpoint inhibitors (ICI) in 11 patients with urothelial carcinoma and 7 patients with renal cell carcinoma who underwent surgery and received ICIs for disease recurrence. Immunohistochemical analyses for CD8, TIA-1, HLA class I, HLA-DR, and PD-L1 were performed and scored 0–3. T-cell infiltration pattern was classified into desert, excluded, partially inflamed, and inflamed. Tumors with an inflamed or partially inflamed pattern and positive scores (score ≥ 1) for all five immune markers were classified as “immune-hot” and others as “immune-cold.” Association between the immunohistogram and ICI treatment efficacy was evaluated with objective response rate, disease control rate (DCR), progression-free survival (PFS), and cancer-specific survival (CSS). Eight (44%) and 10 (56%) patients had immune-hot and immune-cold tumors, respectively. Immune-hot tumors showed a higher DCR (100% vs. 40%, p  < 0.01), longer PFS (median unreached for hot, 1.3 months for cold, p  < 0.01), and longer CSS (median unreached for hot, 3.3 months for cold, p  < 0.01) than immune-cold tumors. The immunohistogram could be clinically useful as an accessible biomarker for precision cancer immunotherapy in urological cancer.

Photodynamic diagnosis/therapy (PDD/PDT) are novel modalities for the diagnosis and treatment of cancer. The photosensitizer protoporphyrin IX is metabolized from 5-aminolevulinic acid (5-ALA) intracellularly, and PDD/PDT using 5-ALA have been approved in dermatologic malignancies and gliomas. However, the molecular mechanism that defines the efficacy of PDD/PDT is unknown. In this study, we analyzed the functions of ATP-binding cassette (ABC) transporters in PDD using 5-ALA. Most of the human gastrointestinal cancer line cells examined showed a homogenous staining pattern with 5-ALA, except for the pancreatic cancer line PANC-1, which showed heterogeneous staining. To analyze this heterogeneous staining pattern, single cell clones were established from PANC-1 cells and the expression of ABC transporters was assessed. Among the ABC transporter genes examined, ABCG2 showed an inverse correlation with the rate of 5-ALA-positive staining. PANC-1 clone #2 cells showed the highest level of ABCG2 expression and the lowest level of 5-ALA staining, with only a 0.6% positive rate. Knockdown of the ABCG2 gene by small interfering RNAs increased the positive rate of 5-ALA staining in PANC-1 wild-type and clone cells. Interestingly, PANC-1 clone #2 cells showed the high sphere-forming ability and tumor-formation ability, indicating that the cells contained high numbers of cancer stem cells (CSCs). Knockdown or inhibition of ABCG2 increased the rate of 5-ALA staining, but did not decrease sphere-forming ability. These results indicate that gastrointestinal cancer cell lines expressing high levels of ABCG2 are enriched with CSCs and show low rates of 5-ALA staining, but 5-ALA staining rates can be improved by inhibition of ABCG2.

Early diagnosis is essential for the safer perinatal management of placenta accreta spectrum (PAS). We used transcriptome analysis to investigate diagnostic maternal serum biomarkers and the mechanisms of PAS development. We analyzed eight formalin-fixed paraffin-embedded placental specimens from two placenta increta and three placenta percreta cases who underwent cesarean hysterectomy at Sapporo Medical University Hospital between 2013 and 2019. Invaded placental regions were isolated from the uterine myometrium and RNA was extracted. The transcriptome difference between normal placenta and PAS was analyzed by microarray analysis. The PAS group showed markedly decreased expression of placenta-specific genes such as LGALS13 and the pregnancy-specific beta-1-glycoprotein (PSG) family. Term enrichment analysis revealed changes in genes related to cellular protein catabolic process, female pregnancy, autophagy, and metabolism of lipids. From the highly dysregulated genes in the PAS group, we investigated the expression of PSG family members, which are secreted into the intervillous space and can be detected in maternal serum from the early stage of pregnancy. The gene expression level of PSG6 in particular was progressively decreased from placenta increta to percreta. The PSG family, especially PSG6, is a potential biomarker for PAS diagnosis.

Immune checkpoint inhibitors (ICIs) have provided an additional treatment option for various types of human cancers. However, ICIs often induce various immune‐related adverse events (irAEs). Enterocolitis is a major irAE with poorly understood histopathological characteristics. In this study, we retrospectively investigated the histopathology of colon tissue samples from 17 patients treated with ICIs. There were two major histological patterns of colitis: an ulcerative colitis‐like pattern and a graft vs host disease‐like pattern. Although these two patterns of colitis were mutually exclusive, both patterns often showed a characteristic that we call “subepithelial surface granulomatosis” (SSG), which has not been reported in other types of colitis. SSG was found even in colon tissue without symptoms or endoscopic findings of colitis. Given the increasing reports of sarcoid reaction or exacerbation of tuberculosis after treatment with ICIs, granuloma formation could be a histological hallmark of systemic immune activation by ICIs. Although statistical significance was not obtained, probably because of the small sample size, SSG may be a surrogate biomarker of systemic anticancer immune activation. We propose that a prospective study with larger sample size be performed. In this study, we retrospectively investigated the histopathology of colon tissue samples from 17 patients treated with immune checkpoint inhibitors. Interestingly, we found characteristic “subepithelial surface granulomatosis” (SSG) in the colon tissue. Although statistical significance was not obtained, probably because of the small sample size, SSG may be a surrogate biomarker of systemic anti‐cancer immune activation.

Recent studies have revealed that treatment-resistant cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) can be targeted by cytotoxic T lymphocytes (CTLs). CTLs recognize antigenic peptides derived from tumor-associated antigens; thus, the identification of tumor-associated antigens expressed by CSCs/CICs is essential. Human leucocyte antigen (HLA) ligandome analysis using mass spectrometry enables the analysis of naturally expressed antigenic peptides; however, HLA ligandome analysis requires a large number of cells and is challenging for CSCs/CICs. In this study, we established a novel bladder CSC/CIC model from a bladder cancer cell line (UM-UC-3 cells) using an ALDEFLUOR assay. CSCs/CICs were isolated as aldehyde dehydrogenase (ALDH)-high cells and several ALDHhigh clone cells were established. ALDHhigh clone cells were enriched with CSCs/CICs by sphere formation and tumorigenicity in immunodeficient mice. HLA ligandome analysis and cap analysis of gene expression using ALDHhigh clone cells revealed a distinctive antigenic peptide repertoire in bladder CSCs/CICs, and we found that a glutamate receptor, ionotropic, kainite 2 (GRIK2)-derived antigenic peptide (LMYDAVHVV) was specifically expressed by CSCs/CICs. A GRIK2 peptide-specific CTL clone recognized GRIK2-overexpressing UM-UC-3 cells and ALDHhigh clone cells, indicating that GRIK2 peptide can be a novel target for bladder CSC/CIC-targeting immunotherapy.

The prognosis of advanced pancreatic adenocarcinoma is still extremely poor. This study sought to determine the efficacy of, and immunological response to, peptide vaccination therapy in patients with this disease. In this multicenter randomized phase II study, patients with advanced pancreatic adenocarcinoma after gemcitabine and/or tegafur/gimeracil/oteracil were randomly assigned to 3 groups that each received a 2‐step treatment course. In Step 1, the groups received treatments of: (i) survivin 2B peptide (SVN‐2B) plus interferon‐β (IFNβ); (ii) SVN‐2B only; or (iii) placebo until the patients show progression. In Step 2, all patients who consented to participate received 4 treatments with SVN‐2B plus IFNβ. The primary endpoint was progression‐free survival (PFS) after initiation of Step 1 treatment. Secondary endpoints included immunological effects assessed by analysis of PBMCs after Step 1. Eighty‐three patients were randomly assigned to receive SVN‐2B plus IFNβ (n = 30), SVN‐2B (n = 34), or placebo (n = 19). No significant improvement in PFS was observed. Survivin 2B‐specific CTLs were found to be increased in the SVN‐2B plus IFNβ group by tetramer assay. Among patients who participated in Step 2, those who had received SVN‐2B plus IFNβ in Step 1 showed better overall survival compared with those who had received placebo in Step 1. Patients vaccinated with SVN‐2B plus IFNβ did not have improved PFS, but showed significant immunological reaction after vaccination. Subgroup analysis suggested that a longer SVN‐2B plus IFNβ vaccination protocol might confer survival benefit. (Clinical trial registration number: UMIN 000012146). Randomized phase II trial of survivin 2B peptide vaccination for patients with HLA‐A24‐positive pancreatic adenocarcinoma is reported. Although peptide vaccination did not improved progression‐free survival, subgroup analysis revealed that longer peptide vaccination might confer survival benefit.

In many malignancies, an increased number of tumor-infiltrating lymphocytes (TILs) is recognized as a favorable prognostic factor, with exceptions such as renal cell carcinoma. However, the clinical significance of TIL size remains unclear. T-cell activation by mitogens increases cell size, partly via c-myc expression, suggesting that larger T cells may be more activated. We hypothesized that TIL size might be prognostically relevant in cancer patients. Here, we examined the relationship between the size and number of tumor-infiltrating CD8 + T cells and patient prognosis in 96 cases of esophageal squamous cell carcinoma (ESCC). We employed artificial intelligence (AI) analysis to quantify the mean size of intratumoral CD8+ T cells in each sample. Patients were then divided into “Large” and “Small” CD8+ T cell groups according to the median T-cell size. Similarly, we classified cases into “High” and “Low” groups based on CD8 + T-cell numbers. We found that patients in the Large CD8+ T cell group had significantly better overall survival than those in the Small CD8+ T cell group by a univariate analysis ( p  = 0.039), but the difference did not reach statistical significance in a multivariate analysis ( p  = 0.054). Patients in the High CD8 + T cell group had better outcomes than those in the Low CD8+ T cell group. There was no significant correlation between CD8+ T cell size and count, and their combination (Large/High) identified a subgroup of patients with the most favorable prognosis. Our findings suggest that CD8+ T cell size could serve as an independent prognostic marker in ESCC.