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The relationship between autophagy and diabetic peripheral neuropathy (DPN) has been highlighted in a only few reports. Using an animal model, we investigated the relationship between autophagy and DPN, and focused particularly on changes in autophagy in Schwann cells.The ultrastructural features of DPN mice were evaluated in vivo using transmission electron microscopy. Dysfunction of autophagy in DPN was evaluated using immunofluorescence microscopy and western blot analysis of autophagy-related proteins, including Beclin1, LC3, and p62. Reactive oxygen species (ROS) levels were measured in vitro in glucose-treated Schwann cells. Dysfunction of autophagy in glucose-treated Schwann cells was examined by immunofluorescence microscopy and western blot analysis.Reduced myelin thickness and axonal shrinkage were observed in the sciatic nerves of DPN mice. ROS levels were increased in high glucose-treated Schwann cells (p0.05). The expression of Becline1 was increased in DPN mice and high glucose-treated Schwann cells (p0.05), whereas the expression of LC3-Ⅱ/LC3-Ⅰ ratio and p62 were decreased in DPN mice and high glucose-treated Schwann cells (p0.05).Our results suggest that increased levels of ROS induced by high glucose may contribute to autophagy dysfunction in Schwann cells. Autophagy dysfunction especially in Schwann cells may be an underlying cause of DPN.

FPKM expression values of 2223 genes deregulated in response to treatment in at least one condition compared to control (P < 0.05)

Supplementary Figure S1. Expression of BCL2L1 and BCL2 in CRC cell lines; Supplementary Figure S2. Estimated BCL2L1 copy numbers of CRC patients by droplet digital PCR (ddPCR); Supplementary Figure S3. Screening of MCL1-modulating agents for synergistic effect with ABT- 263 in KRAS-mutated and BCL2L1-amplified CRC cells (SW620); Supplementary Figure S4. Screening of MCL1-modulating agents for synergistic effect with ABT- 263 in KRAS wild and BCL2L1-diploid CRC cells (SNU1684); Supplementary Figure S5. Effect of ABT-263 and YM-155 combination treatment in CRC cell lines with different KRAS/BRAF mutation status and BCL2L1 copy numbers; Supplementary Figure S6. Effect of ABT-263 and YM-155 in siBCL2L1-treated SW620 cells; Supplementary Figure S7. The quantification of MCL1 protein expression and levels of cleaved caspase 3 and cleaved PARP from Figure 2; Supplementary Figure S8. The quantification of MCL1 protein expression and levels of cleaved caspase 3 and cleaved PARP from Supplementary Figure S5; Supplementary Figure S9. Estimation of apoptosis induced by combination of ABT-263 and YM- 155; Supplementary Figure S10. The quantification of protein expression levels from Figure 3; Supplementary Figure S11. The quantification of protein expression levels from Figure 4; Supplementary Figure S12. The quantification of protein expression levels from Figure 5; Supplementary Figure S13. In vivo efficacy of ABT-263 and YM-155 treatment in CRC cell line xenograft models.

Purpose:Genomic and transcriptomic alterations during metastasis are considered to affect clinical outcome of colorectal cancers, but detailed clinical implications of metastatic alterations are not fully uncovered. We aimed to investigate the effect of metastatic evolution on in vivo treatment outcome, and identify genomic and transcriptomic alterations associated with drug responsiveness.Experimental Design:We developed and analyzed patient-derived xenograft (PDX) models from 35 patients with colorectal cancer including 5 patients with multiple organ metastases (MOMs). We performed whole-exome, DNA methylation, and RNA sequencing for patient and PDX tumors. With samples from patients with MOMs, we conducted phylogenetic and subclonal analysis and in vivo drug efficacy test on the corresponding PDX models.Results:Phylogenetic analysis using mutation, expression, and DNA methylation data in patients with MOMs showed that mutational alterations were closely connected with transcriptomic and epigenomic changes during the tumor evolution. Subclonal analysis revealed that initial primary tumors with larger number of subclones exhibited more dynamic changes in subclonal architecture according to metastasis, and loco-regional and distant metastases occurred in a parallel or independent fashion. The PDX models from MOMs demonstrated therapeutic heterogeneity for targeted treatment, due to subclonal acquisition of additional mutations or transcriptomic activation of bypass signaling pathway during tumor evolution.Conclusions:This study demonstrated in vivo therapeutic heterogeneity of colorectal cancers using PDX models, and suggests that acquired subclonal alterations in mutations or gene expression profiles during tumor metastatic processes can be associated with the development of drug resistance and therapeutic heterogeneity of colorectal cancers.

Supplementary Materials and Methods, Supplementary Figures S1-S10 and Supplementary Tables S1-S6

We experimentally demonstrated V-groove-based self-aligned SiN edge coupler (EC) on a monolithic CMOS-SiPh platform. 520 mW power handling capability were achieved.

Purpose: We performed a prospective study to assess whether the use adhesion barrier Interguard® reduces the rate of adhesion grade in patients who undergoing colorectal surgery plus temporary loop ileostomy as compared to that of the no additive control group.Methods: A total of 38 patients were enrolled between May 2019 and July 2020.Among the 38 enrolled patients, 14 patients received Interguard®, and 24 patients received no additive.Results There was no significant difference of the baseline characteristics between each group. There were no significant differences between the groups for the type of operation (P = 0.163). Operation time was not significantly different between the two groups (P = 0.471). The blood laboratory data was not significantly different from day 5 and 7. None of the patients experienced wound infection and/or anastomotic leakage. None of the patients in Interguard® group developed small bowel ileus after the ileostomy takedown. Adhesion score less than 5 was significantly lower in Interguard® group as compared to control group (P = 0.001). Lower abdomen adhesion score was significantly higher in control group (P = 0.009)ConclusionsThe application of Interguard® did reduce the adhesion grade in patients who were undergoing colorectal surgery and temporary ileostomy as compared to the control group.

Background: PI3Kδ and PI3Kγ are mainly expressed in leukocytes, indicating that they are involved in immune modulation of the tumor microenvironment (TME). Additionally, DNA-dependent protein kinase (DNA-PK) plays a pivotal role as a mediator in repairing DNA damage in cancer cells. Here, we developed a triple inhibitor of PI3Kδ/γ and DNA-PK, BR101801, to promote antitumor immune responses and thereby enhance therapeutic efficacy of the PD-1 immune checkpoint inhibitor. Methods: CT26 and MC38 colon tumor-bearing immunocompetent mice were treated with BR101801 (50 mg/kg, PO) daily and/or anti-PD-1 (8 mg/kg, IP) twice a week. The tumors were comprehensively analyzed using flow cytometry, multiplex tissue imaging, and NanoString profiling methods. Results: Oral administration of BR101801 suppressed tumor growth without significant toxicities. The optimal schedule for BR101801 was determined by activation of T cell immunity in the TME. BR101801 monotherapy significantly increased CD8+ cytotoxic T cells and decreased CD4+CD25+Foxp3+ regulatory T cells, while no changes were observed in tumor-associated macrophages. Moreover, BR101801 increased AH-1+ tumor-specific CD8+ T cells within BR101801-treated CT26 tumors. BR101801 induced extensive immune remodeling of the TME by altering immune-related genes. Notably, gene signatures related to DNA damage and inflammatory responses were more enriched in BR101801-treated tumors. Furthermore, the combination immunotherapy of BR101801 and anti-PD-1 antibody strongly suppressed tumor growth and improved antitumor immunity within the TME, leading to complete tumor regression. Conclusion: Our study demonstrates that BR101801, an oral triple inhibitor of PI3Kδ/γ and DNA-PK, effectively elicited anticancer immune responses within the TME and potently inhibited tumor progression in combination with immune checkpoint blockade. Citation Format: Seung Joon Lee, Won Suk Lee, Eun-Jin Go, Hannah Yang, Hong Ro Kim, Bong-Seog Kim, Chan Kim, Hong Jae Chon. An oral triple inhibitor of PI3Kδ/γ and DNA-PK elicits anticancer immunity and potentiates therapeutic efficacy of immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1800.

The dynamic behaviors of brain glial cells in various neuroinflammatory conditions and neurological disorders have been reported; however, little is known about the underlying intracellular signaling pathways. Here, we developed a multiplexed kinome-wide siRNA screen to identify the kinases regulating several inflammatory phenotypes of mouse glial cells in culture, including inflammatory activation, migration, and phagocytosis of glia. Subsequent proof-of-concept experiments involving genetic and pharmacological inhibitions indicated the importance of T-cell receptor signaling components in microglial activation and a metabolic shift from glycolysis to oxidative phosphorylation in astrocyte migration. This time- and cost-effective multiplexed kinome siRNA screen efficiently provides exploitable drug targets and novel insight into the mechanisms underlying the phenotypic regulation of glial cells and neuroinflammation. Moreover, the kinases identified in this screen may be relevant in other inflammatory diseases and cancer, wherein kinases play a critical role in disease signaling pathways.

596 Background: We elucidated the clinical and immunologic implications of serum interleukin (IL)-6 levels in patients with unresectable hepatocellular carcinoma (HCC) treated with atezolizumab and bevacizumab (Ate/Bev). Methods: We prospectively enrolled 165 patients with unresectable HCC (Discovery cohort: 84 patients from three centers; Validation cohort: 81 patients from one center). Baseline blood samples were analyzed using a flow cytometric bead array. The tumor immune microenvironment was analyzed using RNA sequencing. Results: In the Discovery cohort, clinical benefit (CB) was defined as a complete or partial response or stable disease for ≥ 6 months. Among various blood-based biomarkers, the serum IL-6 level was significantly higher in patients without CB than in those with CB (mean 11.56 vs. 5.05 pg/mL, P=0.02). The optimal cutoff value for high IL-6 was determined as 18.49 pg/mL using maximally selected rank statistics, and 15.2% of patients were identified to have high IL-6 levels at baseline. In both the Discovery and Validation cohorts, patients with high baseline IL-6 levels had a reduced response rate and worse progression-free and overall survival after Ate/Bev compared with that of patients with low IL-6 levels. In the multivariable Cox regression analysis, the clinical implication of high IL-6 levels persisted even after adjustment for various confounding factors. Patients with high IL-6 levels showed reduced interferon-γ and tumor necrosis factor-α secretion from CD8+ T cells. Moreover, excess IL-6 suppressed cytokine production and CD8+ T-cell proliferation. Finally, patients with high IL-6 levels exhibited a non-T-cell-inflamed immunosuppressive tumor microenvironment. Conclusions: High baseline IL-6 levels can be associated with poor clinical outcomes and impaired T-cell function in patients with unresectable HCC after Ate/Bev treatment.

Strawberry is ranked third in the value of production of the crops in Florida, USA. Classifying strawberry maturity and monitoring strawberry growth status in the field is very critical for accurate strawberry yield prediction, efficient strawberry field management, and achieving the highest crop production. The traditional method of distinguishing strawberry maturity is based on either physical appearance or internal chemical substance content. However, the traditional method is time-consuming and costly. In this research, an automatic strawberry maturity classification system was developed for the rapid and accurate classification of different strawberry maturity stages. A state-of-the-art deep learning method, You Only Look Once (YOLOv3), which is good at small object detection, was trained and applied to detect strawberry flowers and strawberry fruit at different maturity stages. Two strawberry image acquisition methods, aerial imaging and near-ground imaging, were compared by using the same deep learning image processing method. As a result, three and seven strawberry maturity stages were classified for unmanned aerial vehicle (UAV) images and near-ground digital camera images, respectively. For UAV images, the highest mean average precision (mAP) of strawberry maturity classification was 0.88 for a test data set at 2 m, and the highest classification average precision (AP) was 0.93 for fully matured fruit. For near-ground digital camera images, the mAP of strawberry maturity classification was 0.89, and the highest classification AP was 0.94 for fully matured fruit as well. The result shows that YOLOv3 is an excellent approach for strawberry maturity classification on both image types.

Renal cell carcinoma (RCC) is the most common type of kidney malignancy worldwide with Pembrolizumab and axitinib treatment (Pembro/Axi) amongst the most effective first-line immunotherapies for advanced RCC. However, it remains difficult to predict treatment response and early resistance. Therefore, we evaluated whether baseline serum interleukin-6 (IL-6) could be a predictive biomarker. Between November 2019 and December 2021, 58 patients with advanced RCC were enrolled, administered first-line Pembro/Axi, and baseline blood samples were analyzed using flow cytometry. The mean baseline serum IL-6 concentration was 8.6 pg/mL in responders and 84.1 pg/mL in patients with progressive disease. The IL-6 cut-off value was set at 6.5 pg/mL using time-dependent receiver operating characteristic curves, with 37.9% of patients having high baseline serum IL-6 levels and 62.1% having low levels. Objective response rates were 58.3% and 36.4% in low and high IL-6 groups, respectively. Overall survival and progression-free survival were longer in patients with low IL-6 levels than in those with high levels. High IL-6 levels were related to reduced interferon-γ and tumor necrosis factor-α production from CD8+ T cells. Overall, high baseline serum IL-6 levels were associated with worse survival outcomes and reduced T-cell responses in Pembro/Axi-treated advanced RCC patients.

ImportanceAdministration of atezolizumab could be immunogenic and induce undesirable antidrug antibody (ADA) responses. This may interfere with atezolizumab-mediated actions, affecting drug clearance and serum concentration or inducing antibody neutralization.ObjectiveTo determine the clinical and immunological associations of highly elevated ADA levels with clinical outcomes after atezolizumab/bevacizumab (Atezo/Bev) treatment in patients with advanced hepatocellular carcinoma (HCC).Design, Setting, and ParticipantsThis cohort study prospectively enrolled 174 patients with advanced HCC treated with first-line Atezo/Bev (discovery cohort: 61 patients from 1 center; validation cohort: 113 patients from 4 centers).ExposuresSerum ADA levels at pretreatment and 3 weeks (cycle 2 day 1 [C2D1]) were analyzed using competitive enzyme-linked immunosorbent assays. In addition, samples were subjected to serological and flow cytometric analyses.Main Outcomes and MeasuresOverall, ADA positivity was associated with treatment outcomes and T-cell functions.ResultsAfter excluding patients with inadequate samples, follow-up loss, or consent withdrawal, 132 patients (discovery cohort: 50 patients; 41 [82.0%] men; median age [IQR], 61 [55-70] years; validation cohort: 82 patients; 70 [85.4%] men; median age [IQR], 61 [53-68] years) were analyzed, and robust ADA (≥1000 ng/mL) responses at C2D1 were identified in 23 (17.4%) of the patients. Patients with progressive disease exhibited higher ADA levels (median [IQR], 65.2 [0-520.4] ng/mL) at C2D1 than in responders (median [IQR], 0 [0-117.5] ng/mL). In both discovery and validation cohorts, patients with high ADA levels at C2D1 were associated with a reduced response rate (discovery cohort: 34% vs 11%; validation cohort: 29% vs. 7%) and worse progression-free survival (discovery cohort: hazard ratio [HR], 2.84; 95% CI, 1.31-6.13; P = .005; validation cohort: HR, 2.52; 95% CI, 1.27-5.01; P = .006) and overall survival (discovery cohort: HR, 3.30; 95% CI, 1.43-7.64; P = .003; validation cohort: HR, 5.81, 95% CI, 2.70-12.50; P = .001) with Atezo/Bev compared with those with low ADA levels. In multivariable Cox regression, the clinical implication of high ADA levels persisted even after adjusting for various confounding factors and was most significant at 1000 ng/mL or greater. Compared with patients with low ADA levels, patients with high ADA levels exhibited reduced serum atezolizumab concentrations, impaired CD8-positive T-cell proliferation, and had decreased interferon-γ and tumor necrosis factor-α from CD8-positive T cells compared with patients with low ADA levels.Conclusions and RelevanceThis cohort study found that highly elevated ADA levels at C2D1 may be associated with poor clinical outcomes in patients with advanced HCC treated with Atezo/Bev. High ADA levels may reduce atezolizumab exposure and attenuate the anticancer efficacy of the drug.

Introduction: Tumor vasculature plays an important role in promoting tumor progression and immune evasion. Previously, we developed PB101, a novel glycosylated decoy receptor that can neutralize VEGF-A, VEGF-B, and PlGF, and demonstrated that it potently inhibits tumor angiogenesis, thereby retarding tumor growth and metastasis. However, its impact on tumor microenvironment (TME), especially anti-cancer immunity, has not been fully elucidated. Here, we investigated distinct modes of immune modulation by PB101 compared with other anti-angiogenic agents, and evaluated its immunotherapeutic potential when combined with immune checkpoint inhibitors (ICIs). Methods: MC38 colon or Hepa-V liver cancer-bearing mice were treated with PB101 or other anti-angiogenic agents with or without various ICIs. The TME was evaluated by histologic, flow cytometric, and Nanostring immune profiling analyses. Results: PB101 markedly suppressed MC38 colon and Hepa-V liver cancer growth in vivo. It simultaneously inhibited both VEGF and PlGF signaling within the TME, thus suppressing tumor angiogenesis. PB101 also induced extensive immune remodeling of the TME. PB101 enhanced intratumoral dendritic cells, but reduced M2-like tumor-associated macrophages. Moreover, PB101 resulted in high influx of activated CD8+, but not CD4+, T cells into the TME. PB101 exhibited the most prominent immune-modulating effect compared with other vascular targeting agents. When combined with various ICIs, PB101 had the greatest synergism with anti-PD-L1 antibody, inducing complete tumor regression and long-term survival. Furthermore, PB101-based combination immunotherapy induced a durable and protective immunity against subcutaneous tumor re-challenge and hematogenous lung metastasis. Conclusion: PB101 elicited strong anti-tumor immunity more effectively than other anti-angiogenic agents. PB101 can strengthen the immunotherapeutic efficacy of PD-L1 inhibitor by establishing a potent and durable immune memory. The combination of PB101 and PD-LI needs to be further validated in clinical trials. Citation Format: Hannah Yang, Seung Joon Lee, Hyun-Gul Yang, Won Suk Lee, Jaekyung Cheon, Beodeul Kang, Sung-Eun Kim, Hyeseong Lim, Hong Jae Chon, Chan Kim. PB101 simultaneously targets tumor angiogenesis and immunity and enhances the efficacy of PD-L1 immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5548.

BackgroundToll-like receptors (TLRs) are critical innate immune sensors that elicit antitumor immune responses in cancer immunotherapy. Although a few TLR agonists have been approved for the treatment of patients with early-stage superficial cancers, their therapeutic efficacy is limited in patient with advanced invasive cancers. Here, we identified the therapeutic role of a TLR2/3 agonist, L-pampo (LP), which promotes antitumor immunity and enhances the immune checkpoint blockade.MethodsWe generated LP by combining a TLR2 agonist, Pam3CSK4, with a TLR3 agonist, Poly (I:C). Immune responses to stimulation with various TLR agonists were compared. Tumor-bearing mice were intratumorally treated with LP, and their tumor sizes were measured. The antitumor effects of LP treatment were determined using flow cytometry, multiplexed imaging, and NanoString nCounter immune profiling. The immunotherapeutic potential of LP in combination with α-programmed cell death protein-1 (PD-1) or α-cytotoxic T-lymphocytes-associated protein 4 (CTLA-4) was evaluated in syngeneic MC38 colon cancer and B16F10 melanoma.ResultsThe LP treatment induced a potent activation of T helper 1 (Th1) and 2 (Th2)-mediated immunity, tumor cell apoptosis, and immunogenic tumor cell death. Intratumoral LP treatment effectively inhibited tumor progression by activating tumor-specific T cell immunity. LP-induced immune responses were mediated by CD8+T cells and interferon-γ, but not by CD4+T cells and CD25+T cells. LP simultaneously activated TLR2 and TLR3 signaling, thereby extensively changing the immune-related gene signatures within the tumor microenvironment (TME). Moreover, intratumoral LP treatment led to systemic abscopal antitumor effects in non-injected distant tumors. Notably, LP treatment combined with ɑPD-1 and ɑCTLA-4 further enhanced the efficacy of monotherapy, resulting in complete tumor regression and prolonged overall survival. Furthermore, LP-based combination immunotherapy elicited durable antitumor immunity with tumor-specific immune memory in colon cancer and melanoma.ConclusionsOur study demonstrated that intratumoral LP treatment improves the innate and adaptive antitumor immunity within the TME and enhances the efficacy of αPD-1 and αCTLA-4 immune checkpoint blockade.