Elimination of residual undifferentiated induced pluripotent stem cells (iPSCs) using irradiation for safe clinical applications of iPSC-derived cardiomyocytes.

A major concern in the clinical application of induced pluripotent stem cells (iPSCs) is the prevention of tumorigenesis after implantation. Stem cells with high proliferative and differentiation potential are sensitive to radiation. Therefore, we hypothesized that irradiation may selectively eliminate residual undifferentiated human iPSCs (hiPSCs) in a cell population containing differentiated cardiomyocytes derived from hiPSCs (hiPSCs-CMs) and thus reduce tumorigenicity in vivo. hiPSC-CMs were irradiated with X-rays, after which the cell proliferation, apoptosis, morphology, and gene expression were analyzed. The gene expression of Lin28A, Nanog, Oct3/4, and SRY-box 2 was significantly lower in the irradiation group than in the control group. Irradiated hiPSC-CMs showed no change in proliferation potency and morphology compared to untreated hiPSC-CMs. Furthermore, irradiation did not induce apoptosis of differentiated cardiomyocytes. No significant difference in the gene expression of cardiac-specific markers, including α-myosin heavy chain, cardiac troponin T, and NK2 Homeobox 5, was observed between the groups. Tumorigenicity tests using NOG mice showed less frequent tumor formation in the irradiation group than in the control group. Irradiation of hiPSC-CMs significantly reduced the number of undifferentiated hiPSC and the tumor formation, while minimizing any adverse effects on hiPSC-CMs, thereby enabling safe hiPSC-based treatment.

Local hyperthermia combined with CTLA-4 blockade induces both local and abscopal effects in a murine breast cancer model.

PURPOSE: To evaluate the antitumor efficacy in local and distant tumors induced by local hyperthermia with CTLA-4 blockade. METHODS: A mouse breast cancer cell line was inoculated into both sides of the legs of mice. The mice were treated with three administrations of CTLA-4 blockade, a single application of local hyperthermia (42.5 °C for 20 min) to the tumor on one side of the leg, or the combination of the two. Tumor growth in locally heated tumors (HT tumors) and unheated distant tumors (UnHT tumors) and overall survival were evaluated. RESULTS: In the combination group, tumor volume significantly decreased for both HT and UnHT tumors compared with the tumors in the untreated and local hyperthermia monotherapy groups. Remarkable efficacy was only observed in the combination therapy group, in which 7 of 18 mice responded to HT and UnHT tumors, with significant prolonged overall survival. CONCLUSIONS: Combination therapy enhanced the antitumor response not only in HT tumors but also in UnHT tumors and prolonged overall survival.

Physical and biological beam modeling for carbon beam scanning at Osaka Heavy Ion Therapy Center.

We have developed physical and biological beam modeling for carbon scanning therapy at the Osaka Heavy Ion Therapy Center (Osaka HIMAK). Carbon beam scanning irradiation is based on continuous carbon beam scanning, which adopts hybrid energy changes using both accelerator energy changes and binary range shifters in the nozzles. The physical dose calculation is based on a triple Gaussian pencil-beam algorithm, and we thus developed a beam modeling method using dose measurements and Monte Carlo simulation for the triple Gaussian. We exploited a biological model based on a conventional linear-quadratic (LQ) model and the photon equivalent dose, without considering the dose dependency of the relative biological effectiveness (RBE), to fully comply with the carbon passive dose distribution using a ridge filter. We extended a passive ridge-filter design method, in which carbon and helium LQ parameters are applied to carbon and fragment isotopes, respectively, to carbon scanning treatment. We then obtained radiation quality data, such as the linear energy transfer (LET) and LQ parameters, by Monte Carlo simulation. The physical dose was verified to agree with measurements to within ±2% for various patterns of volume irradiation. Furthermore, the RBE in the middle of a spread-out Bragg peak (SOBP) reproduced that from passive dose distribution results to within ±1.5%. The developed carbon beam modeling and dose calculation program was successfully applied in clinical use at Osaka HIMAK.

Pitavastatin slows tumor progression and alters urine-derived volatile organic compounds through the mevalonate pathway.

Bone is a frequent site of metastasis from breast cancer, and a desirable drug could suppress tumor growth as well as metastasis-linked bone loss. Currently, no drug is able to cure breast cancer-associated bone metastasis. In this study, we focused on statins that are known to inhibit cholesterol production and act as antitumor agents. After an initial potency screening of 7 U.S. Food and Drug Administration-approved statins, we examined pitavastatin as a drug candidate for inhibiting tumor and tumor-induced bone loss. In vitro analysis revealed that pitavastatin acted as an inhibitor of tumor progression by altering stress to the endoplasmic reticulum, down-regulating peroxisome proliferator-activated receptor γ, and reducing Snail and matrix metalloproteinase 9. In bone homeostasis, it blocked osteoclast development by suppressing transcription factors c-Fos and JunB, but stimulated osteoblast mineralization by regulating bone morphogenetic protein 2 and p53. In a mouse model, pitavastatin presented a dual role in tumor inhibition in the mammary fat pad, as well as in bone protection in the osteolytic tibia. In mass spectrometry-based analysis, volatile organic compounds (VOCs) that were linked to lipid metabolism and cholesterol synthesis were elevated in mice from the tumor-grown placebo group. Notably, pitavastatin-treated mice reduced specific VOCs that are linked to lipid metabolites in the mevalonate pathway. Collectively, the results lay a foundation for further investigation of pitavastatin's therapeutic efficacy in tumor-induced bone loss, as well as VOC-based diagnosis of tumor progression and treatment efficacy.-Wang, L., Wang, Y., Chen, A., Teli, M., Kondo, R., Jalali, A., Fan, Y., Liu, S., Zhao, X., Siegel, A., Minami, K., Agarwal, M., Li, B.-Y., Yokota, H. Pitavastatin slows tumor progression and alters urine-derived volatile organic compounds through the mevalonate pathway.

Finite-element analysis of the mouse proximal ulna in response to elbow loading.

Bone is a mechano-sensitive tissue that alters its structure and properties in response to mechanical loading. We have previously shown that application of lateral dynamic loads to a synovial joint, such as the knee and elbow, suppresses degradation of cartilage and prevents bone loss in arthritis and postmenopausal mouse models, respectively. While loading effects on pathophysiology have been reported, mechanical effects on the loaded joint are not fully understood. Because the direction of joint loading is non-axial, not commonly observed in daily activities, strain distributions in the laterally loaded joint are of great interest. Using elbow loading, we herein characterized mechanical responses in the loaded ulna focusing on the distribution of compressive strain. In response to 1-N peak-to-peak loads, which elevate bone mineral density and bone volume in the proximal ulna in vivo, we conducted finite-element analysis and evaluated strain magnitude in three loading conditions. The results revealed that strain of ~ 1000 µstrain (equivalent to 0.1% compression) or above was observed in the limited region near the loading site, indicating that the minimum effective strain for bone formation is smaller with elbow loading than axial loading. Calcein staining indicated that elbow loading increased bone formation in the regions predicted to undergo higher strain.

X-ray-enhanced cancer cell migration requires the linker of nucleoskeleton and cytoskeleton complex.

The linker of nucleoskeleton and cytoskeleton (LINC) complex is a multifunctional protein complex that is involved in various processes at the nuclear envelope, including nuclear migration, mechanotransduction, chromatin tethering and DNA damage response. We recently showed that a nuclear envelope protein, Sad1 and UNC84 domain protein 1 (SUN1), a component of the LINC complex, has a critical function in cell migration. Although ionizing radiation activates cell migration and invasion in vivo and in vitro, the underlying molecular mechanism remains unknown. Here, we examined the involvement of the LINC complex in radiation-enhanced cell migration and invasion. A sublethal dose of X-ray radiation promoted human breast cancer MDA-MB-231 cell migration and invasion, whereas carbon ion beam radiation suppressed these processes in a dose-dependent manner. Depletion of SUN1 and SUN2 significantly suppressed X-ray-enhanced cell migration and invasion. Moreover, depletion or overexpression of each SUN1 splicing variant revealed that SUN1_888 containing 888 amino acids of SUN1 but not SUN1_916 was required for X-ray-enhanced migration and invasion. In addition, the results suggested that X-ray irradiation affected the expression level of SUN1 splicing variants and a SUN protein binding partner, nesprins. Taken together, our findings supported that the LINC complex contributed to photon-enhanced cell migration and invasion.

P18: Novel Anticancer Peptide from Induced Tumor-Suppressing Cells Targeting Breast Cancer and Bone Metastasis.

BACKGROUND: The skeletal system is a common site for metastasis from breast cancer. In our prior work, we developed induced tumor-suppressing cells (iTSCs) capable of secreting a set of tumor-suppressing proteins. In this study, we examined the possibility of identifying anticancer peptides (ACPs) from trypsin-digested protein fragments derived from iTSC proteomes. METHODS: The efficacy of ACPs was examined using an MTT-based cell viability assay, a Scratch-based motility assay, an EdU-based proliferation assay, and a transwell invasion assay. To evaluate the mechanism of inhibitory action, a fluorescence resonance energy transfer (FRET)-based GTPase activity assay and a molecular docking analysis were conducted. The efficacy of ACPs was also tested using an ex vivo cancer tissue assay and a bone microenvironment assay. RESULTS: Among the 12 ACP candidates, P18 (TDYMVGSYGPR) demonstrated the most effective anticancer activity. P18 was derived from Arhgdia, a Rho GDP dissociation inhibitor alpha, and exhibited inhibitory effects on the viability, migration, and invasion of breast cancer cells. It also hindered the GTPase activity of RhoA and Cdc42 and downregulated the expression of oncoproteins such as Snail and Src. The inhibitory impact of P18 was additive when it was combined with chemotherapeutic drugs such as Cisplatin and Taxol in both breast cancer cells and patient-derived tissues. P18 had no inhibitory effect on mesenchymal stem cells but suppressed the maturation of RANKL-stimulated osteoclasts and mitigated the bone loss associated with breast cancer. Furthermore, the P18 analog modified by N-terminal acetylation and C-terminal amidation (Ac-P18-NH2) exhibited stronger tumor-suppressor effects. CONCLUSIONS: This study introduced a unique methodology for selecting an effective ACP from the iTSC secretome. P18 holds promise for the treatment of breast cancer and the prevention of bone destruction by regulating GTPase signaling.

Synergistic Effects of Melatonin on Radiosensitization in Carbon-ion Radiotherapy.

BACKGROUND/AIM: Despite the established antitumor effectiveness and synergistic interactions of melatonin with photon irradiation, its role in carbon-ion radiotherapy remains uncertain. This study aimed to elucidate the mechanisms and potential clinical advantages of combining exogenous melatonin therapy with carbon-ion radiotherapy. MATERIALS AND METHODS: The investigation assessed the impact of combining exogenous melatonin with photon or carbon-ion irradiation on cell-cycle modulation and DNA-repair capability using the melanoma cell line B16F10. RNA sequencing and bioinformatics analysis were conducted to explore mechanisms and evaluate potential clinical benefits, with validation performed on the osteosarcoma cell line LM8. RESULTS: Pre-treatment with melatonin reduced the survival fraction of B16F10 and LM8 cells upon exposure to photon and carbon-ion radiation. Mechanistically, melatonin was found to inhibit G2/M arrest, preserve DNA damage, and suppress key genes involved in DNA double-strand break repair after 8 Gy carbon-ion radiation. Furthermore, RNA sequencing and bioinformatics analysis revealed favorable changes in genes associated with survival and metastasis, highlighting potential clinical significance. LM8 cells treated with melatonin exhibited increased radiosensitivity and suppression of DNA-repair proteins. CONCLUSION: The combination of exogenous melatonin not only heightened radiosensitivity and modulated hallmark tumor gene sets in vitro but also markedly suppressed the efficiency of DNA double-strand break-repair pathway, thus enhancing the cytotoxicity of carbon-ion radiotherapy.

Irradiated Cell-derived Exosomes Enhance Cell Proliferation and Radioresistance via the MAPK/Erk Pathway.

BACKGROUND/AIM: Radiation therapy is pivotal in cancer treatment; however, its efficacy is limited by challenges such as tumor recurrence. This study delves into the role of exosomes, which are molecular cargo-bearing vesicles, in influencing cell proliferation, radioresistance, and consequent post-irradiation tumor recurrence. Given the significance of exosomes from irradiated malignancies in diagnostics and therapy, it is vital to delineate their functional dynamics, especially in breast and cervical cancer cell lines, where the impact of irradiation on exosome behavior remains enigmatic. MATERIALS AND METHODS: Using MDA-MB-231 and HeLa cell lines, exosomes were isolated from the culture supernatant via ultracentrifugation. The bicinchoninic acid assay was used to measure exosome quantities in irradiated and non-irradiated cells. Radiosensitivity was assessed using colony formation assays, while the role of the MAPK/Erk signaling pathway in recipient cell proliferation and radioresistance was probed using western blotting. RESULTS: Irradiated cells, in both MDA-MB-231 and HeLa lines, produced significantly more exosomes than their non-irradiated counterparts. Co-culturing irradiated cells with exosomes led to increased cell survival post-irradiation and enhanced cell proliferation in both cell lines. Western blotting indicated elevated p-Erk expression in such cells, underscoring the influence of the MAPK/Erk pathway in radioresistance and proliferation. CONCLUSION: The study establishes a potential nexus between exosome secretion and tumor resurgence following radiotherapy. The spotlight falls on the MAPK/ERK signaling conduit as a key influencer. This new knowledge provides an innovative strategy for counteracting cancer recurrence after radiotherapy, emphasizing the importance of understanding the multifaceted roles of exosomes in this context.

Circulating Plasma Exosomal PD-L1 Predicts Prognosis of Head and Neck Squamous Cell Carcinoma After Radiation Therapy.

Purpose: Radiation therapy is widely used to treat head and neck squamous cell carcinoma (HNSCC). This study evaluated the association between circulating plasma programmed death-ligand 1 (PD-L1) and the outcomes of patients with HNSCC after radiation therapy. Methods and Materials: In this retrospective observational study, plasma samples of 76 patients with HNSCC who underwent radiation therapy from June 2019 to August 2021 were analyzed. These plasma samples were obtained before radiation therapy. The median follow-up was 32.5 months. Total and exosomal PD-L1 was measured by enzyme-linked immunosorbent assay and retrospectively analyzed for association with overall survival (OS), progression-free survival (PFS), and local control (LC). Prognostic factors among patients' characteristics and circulating PD-L1 in plasma were evaluated by univariate (log-rank test) and multivariate (Cox proportional hazards model) analyses. Results: The median concentration of total PD-L1 in plasma was 115.1 pg/mL (95% CI, 114.7-137.9 pg/mL), and the median concentration of exosomal PD-L1 was 2.8 pg/mL (95% CI, 6.0-13.0 pg/mL). Univariate and multivariate analyses showed exosomal PD-L1 as a prognostic factor for PFS and LC. Patients with high exosomal PD-L1 in plasma had poor PFS and LC compared with those with low exosomal PD-L1, indicating that 1-year PFS was 79.2% versus 33.3% (P < .001) and 1-year LC was 87.3% versus 50.0% (P < .001) in patients with high and low exosomal PD-L1, respectively. However, exosomal PD-L1 in plasma had no significant effect on OS. Total PD-L1 in plasma did not correlate with PFS, LC, and OS. Conclusions: The pretreatment circulating exosomal PD-L1 in plasma of patients with HNSCC was a prognostic factor after radiation therapy.

The linker of nucleoskeleton and cytoskeleton complex is required for X-ray-induced epithelial-mesenchymal transition.

The linker of nucleoskeleton and cytoskeleton (LINC) complex has been implicated in various functions of the nuclear envelope, including nuclear migration, mechanotransduction and DNA repair. We previously revealed that the LINC complex component Sad1 and UNC84 domain containing 1 (SUN1) is required for sublethal-dose X-ray-enhanced cell migration and invasion. This study focused on epithelial-mesenchymal transition (EMT), which contributes to cell migration. Hence, the present study aimed to examine whether sublethal-dose X-irradiation induces EMT and whether LINC complex component SUN1 is involved in low-dose X-ray-induced EMT. This study showed that low-dose (0.5 Gy or 2 Gy) X-irradiation induced EMT in human breast cancer MDA-MB-231 cells. Additionally, X-irradiation increased the expression of SUN1. Therefore, SUN1 was depleted using siRNA. In SUN1-depleted cells, low-dose X-irradiation did not induce EMT. In addition, although the SUN1 splicing variant SUN1_916-depleted cells (containing 916 amino acids [AA] of SUN1) were induced EMT by low-dose X-irradiation like as non-transfected control cells, SUN1_888-depleted cells (which encodes 888 AA) were not induced EMT by low-dose X-irradiation. Moreover, since the Wnt/ß-catenin signaling pathway regulates E-cadherin expression via the expression of the E-cadherin repressor Snail, the expression of ß-catenin after X-irradiation was examined. After 24 hours of irradiation, ß-catenin expression increased in non-transfected cells or SUN1_916-depleted cells, whereas ß-catenin expression remained unchanged and did not increase in SUN1- or SUN1_888-depleted cells. Therefore, in this study, we found that low-dose X-irradiation induces EMT, and LINC complex component SUN1, especially SUN1_888, is required for X-ray-induced EMT via activation of the Wnt/ß-catenin signaling pathway.

Treatment planning of carbon ion radiotherapy for prostate cancer based on cellular experiments with PC3 human prostate cancer cells.

[Purpose] Treatment plans for carbon ion radiotherapy (CIRT) in Japan are designed to uniformly deliver the prescribed clinical dose based on the radiosensitivity of human salivary gland (HSG) cells to the planning target volume (PTV). However, sensitivity to carbon beams varies between cell lines, that is, it should be checked that the clinical dose distribution based on the cell radiosensitivity of the treatment site is uniform within the PTV. [Methods] We modeled the linear energy transfer (LET) dependence of the linear-quadratic (LQ) coefficients specific to prostate cancer, which accounts for the majority of CIRT. This was achieved by irradiating prostate cancer cells (PC3) with X-rays from a 4 MV-Linac and carbon beams with different LETs of 11.1-214.3 keV/µm. By using the radiosensitivity of PC3 cells derived from cellular experiments, we reconstructed prostate-cancer-specific clinical dose distributions on patient computed tomography (CT). [Results] The LQ coefficient, α, of PC3 cells was larger than that of HSG cells at low (<50 keV/µm) LET and smaller at high (>50 keV/µm) LET, which was validated by cellular experiments performed on rectangular SOBPs. The reconstructed dose distribution on patient CT was sloped when 1 fraction incident from the one side of the patient was considered, but remained uniform from the sum of 12 fractions of the left-right opposing beams (as is used in clinical practice). [Conclusion] Our study reveals the inhomogeneity of clinical doses in single-field plans calculated using the PC3 radiosensitivity data. However, this inhomogeneity is compensated by using the combination of left-right opposing beams.

KRT13 is upregulated in pancreatic cancer stem-like cells and associated with radioresistance.

Pancreatic cancer is one of the most aggressive cancers and the seventh leading cause of cancer-associated death in the world. Radiation is performed as an adjuvant therapy as well as anti-cancer drugs. Because cancer stem-like cells (CSCs) are considered to be radioresistant and cause recurrence and metastasis, understanding their properties is required for the development of novel therapeutic strategies. To investigate the CSC properties of pancreatic cancer cells, we used a pancreatic CSC model, degron (++) cells, which have low proteasome activity. Degron (++) cells displayed radioresistance in comparison with control cells. Using Ribonucleic acid (RNA) sequencing, we successfully identified KRT13 as a candidate gene responsible for radioresistance. Knockdown of KRT13 sensitized the degron (++) cells to radiation. Furthermore, a database search revealed that KRT13 is upregulated in pancreatic cancer cell lines and that high expression of KRT13 is associated with poorer prognosis. These results indicate that a combination therapy of KRT13 knockdown and radiation could hold therapeutic promise in pancreatic cancer.

The inhibition of pancreatic cancer progression by K-Ras-overexpressing mesenchymal stem cell-derived secretomes.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor survival. To explore an uncharted function of K-Ras proto-oncogene, K-Ras was activated in mesenchymal stem cells (MSCs) and the effects of MSC conditioned medium (CM) on PDAC were examined. Overexpression of K-Ras elevated PI3K signaling in MSCs, and K-Ras/PI3K-activated MSC-derived CM reduced the proliferation and migration of tumor cells, as well as the growth of ex vivo freshly isolated human PDAC cultures. CM's anti-tumor capability was additive with Gemcitabine, a commonly used chemotherapeutic drug in the treatment of PDAC. The systemic administration of CM in a mouse model suppressed the colonization of PDAC in the lung. MSC CM was enriched with Moesin (MSN), which acted as an extracellular tumor-suppressing protein by interacting with CD44. Tumor-suppressive CM was also generated by PKA-activated peripheral blood mononuclear cells. Collectively, this study demonstrated that MSC CM can be engineered to act as a tumor-suppressive agent by activating K-Ras and PI3K, and the MSN-CD44 regulatory axis is in part responsible for this potential unconventional option in the treatment of PDAC.

Proteomes from AMPK-inhibited peripheral blood mononuclear cells suppress the progression of breast cancer and bone metastasis.

Background: During a developmental process, embryos employ varying tactics to remove unwanted cells. Using a procedure analogous to some of the embryonic cells, we generated a tumor-eliminating conditioned medium (CM) from AMPK-inhibited lymphocytes and monocytes in peripheral blood mononuclear cells (PBMCs). Methods: AMPK signaling was inhibited by the application of a pharmacological agent, Dorsomorphin, and the therapeutic effects of their conditioned medium (CM) were evaluated using in vitro cell cultures, ex vivo breast cancer tissues, and a mouse model of mammary tumors and tumor-induced osteolysis. The regulatory mechanism was evaluated using mass spectrometry-based proteomics, Western blotting, immunoprecipitation, gene overexpression, and RNA interference. Results: While AMPK signaling acted mostly anti-tumorigenic, we paradoxically inhibited it to build induced tumor-suppressing cells and their tumor-eliminating CM. In a mouse model of breast cancer, the application of AMPK-inhibited lymphocyte-derived CM reduced mammary tumors additively to a chemotherapeutic agent, Taxol. It also prevented bone loss in the tumor-bearing tibia. Furthermore, the application of CM from the patient-derived peripheral blood diminished ex vivo breast cancer tissues isolated from the same patients. Notably, proteins enriched in CM included Moesin (MSN), Enolase 1 (ENO1), and polyA-binding protein 1 (PABPC1), which are considered tumorigenic in many types of cancer. The tumor-suppressing actions of MSN and ENO1 were at least in part mediated by Metadherin (Mtdh), which is known to promote metastatic seeding. Conclusion: We demonstrated that PBMCs can be used to generate tumor-suppressive proteomes, and extracellular tumor-suppressing proteins such as MSN, ENO1, and PABPC1 are converted from tumor-promoting factors inside cancer cells. The results support the possibility of developing autologous blood-based therapy, in which tumor-suppressing proteins are enriched in engineered PBMC-derived CM by the inhibition of AMPK signaling.

Exploring the Tumor-Suppressing Potential of PSCA in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with low survival rates. We explored an innovative therapeutic approach by leveraging prognostic oncogenic markers. Instead of inhibiting these marker genes, we harnessed their tumor-modifying potential in the extracellular domain. Surprisingly, many of the proteins highly expressed in PDAC, which is linked to poor survival, exhibited tumor-suppressing qualities in the extracellular environment. For instance, prostate stem cell antigens (PSCA), associated with reduced survival, acted as tumor suppressors when introduced extracellularly. We performed in vitro assays to assess the proliferation and migration and evaluated the tumor-modifying capacity of extracellular factors from peripheral blood mononuclear cells (PBMCs) in PDAC tissues. Molecular docking analysis, immunoprecipitation, Western blotting, and RNA interference were employed to study the regulatory mechanism. Extracellular PSCA recombinant protein notably curtailed the viability, motility, and transwell invasion of PDAC cells. Its anti-PDAC effects were partially mediated by Mesothelin (MSLN), another highly expressed tumor-associated antigen in PDAC. The anti-tumor effects of extracellular PSCA complemented those of chemotherapeutic agents like Irinotecan, 5-Fluorouracil, and Oxaliplatin. PSCA expression increased in a conditioned medium derived from PBMCs and T lymphocytes. This study unveils the paradoxical anti-PDAC potential of PSCA, hinting at the dual roles of oncoproteins like PSCA in PDAC suppression.

Suppression of osteosarcoma progression by engineered lymphocyte-derived proteomes.

Cancer cells tend to develop resistance to chemotherapy and enhance aggressiveness. A counterintuitive approach is to tame aggressiveness by an agent that acts opposite to chemotherapeutic agents. Based on this strategy, induced tumor-suppressing cells (iTSCs) have been generated from tumor cells and mesenchymal stem cells. Here, we examined the possibility of generating iTSCs from lymphocytes by activating PKA signaling for suppressing the progression of osteosarcoma (OS). While lymphocyte-derived CM did not present anti-tumor capabilities, the activation of PKA converted them into iTSCs. Inhibiting PKA conversely generated tumor-promotive secretomes. In a mouse model, PKA-activated CM suppressed tumor-induced bone destruction. Proteomics analysis revealed that moesin (MSN) and calreticulin (Calr), which are highly expressed intracellular proteins in many cancers, were enriched in PKA-activated CM, and they acted as extracellular tumor suppressors through CD44, CD47, and CD91. The study presented a unique option for cancer treatment by generating iTSCs that secret tumor-suppressive proteins such as MSN and Calr. We envision that identifying these tumor suppressors and predicting their binding partners such as CD44, which is an FDA-approved oncogenic target to be inhibited, may contribute to developing targeted protein therapy.

Validation of robust radiobiological optimization algorithms based on the mixed beam model for intensity-modulated carbon-ion therapy.

Currently, treatment planning systems (TPSs) that can compute the intensities of intensity-modulated carbon-ion therapy (IMCT) using scanned carbon-ion beams are limited. In the present study, the computational efficacy of the newly designed IMCT algorithms was analyzed for the first time based on the mixed beam model with respect to the physical and biological doses; moreover, the validity and effectiveness of the robust radiobiological optimization were verified. A dose calculation engine was independently generated to validate a clinical dose determined in the TPS. A biological assay was performed using the HSGc-C5 cell line to validate the calculated surviving fraction (SF). Both spot control (SC) and voxel-wise worst-case scenario (WC) algorithms were employed for robust radiobiological optimization followed by their application in a Radiation Therapy Oncology Group benchmark phantom under homogeneous and heterogeneous conditions and a clinical case for range and position errors. Importantly, for the first time, both SC and WC algorithms were implemented in the integrated TPS platform that can compute the intensities of IMCT using scanned carbon-ion beams for robust radiobiological optimization. For assessing the robustness, the difference between the maximum and minimum values of a dose-volume histogram index in the examined error scenarios was considered as a robustness index. The relative biological effectiveness (RBE) determined by the independent dose calculation engine exhibited a -0.6% difference compared with the RBE defined by the TPS at the isocenter, whereas the measured and the calculated SF were similar. Regardless of the objects, compared with the conventional IMCT, the robust radiobiological optimization enhanced the sensitivity of the examined error scenarios by up to 19% for the robustness index. The computational efficacy of the novel IMCT algorithms was verified according to the mixed beam model with respect to the physical and biological doses. The robust radiobiological optimizations lowered the impact of range and position uncertainties considerably in the examined scenarios. The robustness of the WC algorithm was more enhanced compared with that of the SC algorithm. Nevertheless, the SC algorithm can be used as an alternative to the WC IMCT algorithm with respect to the computational cost.

Ultra-high Dose-rate Carbon-ion Scanning Beam With a Compact Medical Synchrotron Contributing to Further Development of FLASH Irradiation.

BACKGROUND/AIM: The focus of this report is establishing an irradiation arrangement to realize an ultra-high dose-rate (uHDR; FLASH) of scanned carbon-ion irradiation possible with a compact commonly available medical synchrotron. MATERIALS AND METHODS: Following adjustments to the operation it became possible to extract ≥1.0×109 carbon ions at 208.3 MeV/u (86 mm in range) per 100 ms. The design takes the utmost care to prevent damage to monitors, particularly in the nozzle, achieved by the uHDR beam not passing through this part of the apparatus. Doses were adjusted by extraction times, using a function generator. After one scan by the carbon-ion beam it became possible to create a field within the extraction time. The Advanced Markus chamber (AMC) and Gafchromic film are then able to measure the absolute dose and field size at a plateau depth, with the operating voltage of the chamber at 400 V at the uHDR for the AMC. RESULTS: The beam scanning utilizing this uHDR irradiation could be confirmed at a dose of 6.5±0.08 Gy (±3% homogeneous) at this volume over at least 16×16 mm2 corresponding to a dose-rate of 92.3 Gy/s (±1.3%). The dose was ca. 0.7, 1.5, 2.9, and 5.4 Gy depending on dose-rate and field size, with the rate of killed cells increasing with the irradiation dose. CONCLUSION: The compact medical synchrotron achieved FLASH dose-rates of >40 Gy/s at different dose levels and in useful field sizes for research with the apparatus and arrangement developed here.