Mouse Subcutaneous Tumor Model Research Articles

Irradiation Induced Activation of cGAS/STING Signaling Promotes Macrophage Anti-Tumor Activity via CXCL9, CXCL10-CXCR3 Axis

<h3>Purpose/Objective(s)</h3> Irradiation induced abundant dsDNA fragments, which can stimulate tumor immune responses by DNA sensing pathway. However, the underlying mechanism, mainly involved immune cells remains unknown. Herein we discovered that macrophage was the main immune cell involved in cGAS/STING DNA sensing pathway induced anti-tumor immune response, and macrophage derived CXCL9, CXCL10 played an important role in infiltration of T cells in tumor via CXCL9, CXCL10-CXCR3 axis. <h3>Materials/Methods</h3> cGAS knockout mice (cGAS-/- mice) and STING-deficient mice were used in the study. Xenograft tumor mouse model was established for in vivo study, using murine hepatoma cell line H22 or ovalbumin (OVA)-expressing H22. Irradiation locally delivered to the tumor area at 8 Gy/day for 3 days using a micro-CT/micro irradiator. <h3>Results</h3> Macrophage was found recruitment to the irradiated tumor site after irradiation. Using C57BL/6J mice bearing xenograft H22 tumors, we found cGAS-/- mice showed higher growth rate after irradiation comparing to tumors growing on wild type mice (WT mice). Using a both sides subcutaneous H22-OVA tumor mouse model, we found a substantial increased OVA-specific CD8+ T cells within the DLN(draining lymph node) of right irradiated tumor of WT mice, but low level within the right DLN of the irradiated tumor of STING-deficient mice and the no-irradiated right tumors of STING-deficient mice or WT mice, which confirmed that cGAS/STING pathway was involved in the radiation-mediated immunomodulation and promoted the radiation-induced adaptive anti-tumor immune activation. We also noted macrophage depletion partial reverse antitumor effect of irradiation in WT tumors, while no significant difference is observed in cGAS^−/− tumor, which revealed macrophage was the main immune cell involved in cGAS/STING DNA sensing pathway induced anti-tumor immune response. Further mechanism study revealed macrophage derived CXCL9, CXCL10 was induced by radiation in a cGAS/STING pathway dependent manner. Intraperitoneal injection of anti-CXCL9, CXCL10 significantly reduce the anti-tumor effect of irradiation in WT mice, but not in STING-deficient mice, these observations suggested macrophage derived CXCL9, CXCL10 were involved in infiltration of T cells in tumor via CXCL9, CXCL10-CXCR3 axis. <h3>Conclusion</h3> cGAS/STING pathway is crucial in the radiation-induced adaptive anti-tumor immune activation. macrophage derived CXCL9, CXCL10, which induced by radiation in a cGAS/STING pathway dependent manner, mediated infiltration of T cells in tumor via CXCL9, CXCL10- CXCR3 axis.

Wnt signaling modulator DKK4 inhibits colorectal cancer metastasis through an AKT/Wnt/β-catenin negative feedback pathway

Aberrant activation of the Wnt/β-catenin signaling pathway is implicated in most malignant cancers, especially in the initiation and progression of colorectal cancer (CRC). DKK4 is a classical inhibitory molecule of the Wnt/β-catenin pathway, but its role in CRC is ambiguous, and the molecular mechanism remains unclear. Here, we determined DKK4 expression was significantly upregulated in 23 CRC cell lines and 229 CRC tissues when analyzed by quantitative PCR and immunohistochemistry, respectively. Our analysis of tissue samples indicated the survival time of CRC patients with high DKK4 expression was longer than that of patients with medium-low DKK4 expression. We examined the effects of DKK4 on cell proliferation and metastasis by cell counting kit-8 assays, transwell assays, and subcutaneous and metastatic mouse tumor models, and we discovered that DKK4 silencing promoted the metastasis of CRC cells both invitro and invivo. Our RNA-seq analysis revealed that AKT2, FZD6, and JUN, which play important roles in AKT and Wnt signaling, were significantly increased after DKK4 knockdown. DKK4 represses Wnt/β-catenin signaling by repressing FZD6 and AKT2/s552 β-catenin in CRC. Further experiments revealed recombinant Wnt3a and LiCl could induce DKK4 expression. Moreover, our bioinformatics analysis and luciferase reporter assays identified posttranscriptional regulators of DKK4 in CRC cells. In summary, DKK4 is elevated in CRC and inhibits cell metastasis by a novel negative feedback mechanism of the Wnt3a/DKK4/AKT/s552 β-catenin regulatory axis to restrict overactivation of Wnt activity in CRC. Therefore, DKK4 restoration may be applied as a potential CRC therapeutic strategy.

OSW-1 induces apoptosis and cyto-protective autophagy, and synergizes with chemotherapy on triple negative breast cancer metastasis.

Triple-negative breast cancer (TNBC) is the most malignant subtype of breast cancer. As yet, chemotherapy with drugs such as doxorubicin is the main treatment strategy. However, drug resistance and dose-dependent toxicities restrict their clinical use. Natural products are major sources of anti-tumor drugs. OSW-1 is a natural compound with strong anti-cancer effects in several types of cancer, but its effects on the efficacy of chemotherapy in TNBC and its underlying mechanism remain unclear. The inhibitory activities of OSW-1 and its combination with several chemotherapy drugs were tested using in vitro assays and in vivo subcutaneous and metastatic mouse TNBC models. The effects of the mono- and combination treatments on TNBC cell viability, apoptosis, autophagy and related signaling pathways were assessed using MTT, flow cytometry, RNA sequencing and immunology-based assays. In addition, the in vivo inhibitory effects of OSW-1 and (combined) chemotherapies were evaluated in subcutaneous and metastatic mouse tumor models. We found that OSW-1 induces Ca2+-dependent mitochondria-dependent intrinsic apoptosis and cyto-protective autophagy through the PI3K-Akt-mTOR pathway in TNBC cells in vitro. We also found that OSW-1 and doxorubicin exhibited strong synergistic anti-TNBC capabilities both in vivo and in vitro. Combination treatment strongly inhibited spontaneous and experimental lung metastases in 4T1 mouse models. In addition, the combination strategy of OSW-1 + Carboplatin + Docetaxel showed an excellent anti-metastatic effect in vivo. Our data revealed the mode of action and molecular mechanism underlying the effect of OSW-1 against TNBC, and provided a useful guidance for improving the sensitivity of TNBC cells to conventional chemotherapeutic drugs, which warrants further investigation.

Berberine enhances the anti-hepatocellular carcinoma effect of NK92-MI cells through inhibiting IFN-gamma-mediated PD-L1 expression

Background and aimsBerberine is one of the most promising clinically tested natural alkaloids, and immunotherapy using natural killer (NK) cells is a potentially effective treatment for hepatocellular carcinoma (HCC). This study aims to elucidate the effect of berberine on the anti-HCC effect of NK92-MI cells. Materials and methodsHuman HCC SMMC-7721 and Hep3B cells were co-incubated with NK92-MI cells, berberine (60 or 80 μmol/L), or their combination for 36 h. To evaluate the killing effect of NK92-MI cells on HCC cells, the release of lactate dehydrogenase (LDH) in HCC cells was measured. The anti-tumor effects of berberine, NK92-MI cells, and their combinations were evaluated by MTS, EdU, Tunel, and Western blot assays. A male BALB/c nude mouse subcutaneous tumor model was used to investigate the anti-HCC effect of berberine and NK92-MI cells in vivo. ResultsThe LDH assay showed that berberine enhanced the cytotoxicity of NK92-MI cells on HCC cells. The combination of berberine and NK92-MI cells demonstrated more obvious anti-HCC effect than did the berberine or NK92-MI single treatment in inhibiting cell proliferation and inducing apoptosis both in vitro and in vivo. Mechanistically, the expression of programmed cell death-ligand 1 (PD-L1) in HCC cells was upregulated after co-culture with NK-92MI cells. PD-L1 expression was knocked down, thereby inhibiting the proliferation and promoting apoptosis of HCC cells, and inhibited by berberine that blocked the secretion of interferon gamma (IFN-γ), thereby enhancing the anti-tumor effect of NK92-MI cells. ConclusionsCurrent data show that the immunomodulatory role of berberine is to enhance the cytotoxic effect of NK92-MI cells and inhibit tumor immune escape by reducing the expression of PD-L1. Our study provides a rationale for the clinical application of berberine in combination with NK cells for the treatment of HCC.

Numerical analysis and animal study of noninvasive handheld electroporation delivery device for skin superficial lesion treatment

Introduction This study aims to investigate the feasibility of a noninvasive handheld electroporation pulses delivery device (EPDD) for electroporation-based treatment (EBT) of skin superficial lesions through numerical analysis and animal study. Methods Finite element analysis was performed to investigate the performance of the EPDD. The electric field, temperature, EI and TI were calculated under pulse voltages of 600, 800, and 1000 V. A mouse subcutaneous tumor model was established to evaluate the performance of the EPDD through histopathology and survival analyses. Results The electrical field strength increased from 151 (600 V) to 252 V/cm (1000 V) in the skin and from 1302 (600 V) to 2171 V/cm (1000 V) in the tumor. The volume of EI grew and reached a plateau at the 165th pulse, whereas the maximum volume of EI increased with higher voltage. The growth tendency of TI differed between groups, and it was higher in the high-voltage group (HVG) than in the low-voltage group. Histopathological analysis showed that the depth and range of the ablation area could be controlled by adjusting pulse voltage. Survival analysis showed that the survival of the HVG was better than that of the low-voltage and the control group (p < 0.01). Conclusions The results demonstrate that the EPDD is feasible, safe, and effective for skin EBT. The volume of EP tissue injury can be controlled by adjusting the pulse voltage, pulse number, and other parameters. The proposed noninvasive handheld EPDD can be a potential therapeutic tool for EBT of superficial skin lesions in the future.

Abstract 5600: Glycopeptides promote anti-cancer immune response against solid tumors

Abstract Only a fraction of patients benefits from immune checkpoint inhibitors (ICI). Tumor cells exhibit a distinct glycosylation pattern and supplementation of glycans has the potential to promote response and overcome resistance to ICI, thus addressing an urgent clinical need. Using a subcutaneous tumor mouse model, we investigated whether two glycopeptide-based test items reduce tumor burden when orally administered alone or in combination with ICI. C57/BL6 mice were supplemented with 3% GNU-201 (n=10) or 3% GNU-101 (n=10) in the drinking water starting 14 days prior to tumor cell injection (d-14), while control mice (n=10) received normal drinking water. On day 0, all mice were injected subcutaneously with YUMM1.7 melanoma cells in each flank. On days 6, 9 and 12, mice in each group received anti-PD1 antibody or an isotype control. Tumor size was measured every 3rd day. On day 15, the mice were sacrificed to analyze tumor weight and tumor infiltrating immune cells. 16S rDNA sequencing of fecal samples was performed on days -14, 0 and 14. GNU-201 and GNU-101 supplementation was well tolerated, no difference in water consumption and no abnormal behavior were detected that would indicate adverse effects of the products. While anti-PD1 on its own had no effect on tumor growth, we observed reduced tumor growth in the GNU-201+anti-PD1, GNU-101 and GNU-101+anti-PD1-treated groups, resulting in a significantly reduced tumor volume in these three groups on day 15. Administration of the two test items had a clear effect on anti-tumor T cell responses. In combination with anti-PD1, GNU-201 promoted the overall abundance of T cells, and among those the proportion of CD8+ T cells and IFNγ producing CD4+ T cells. Even in absence of anti-PD1, GNU-201 promoted the number of IFNγ+ CD8+ T cells, indicating that GNU-201 did not only promote tumor infiltration but also activation of CD8+ T cells, which are known to directly kill tumor cells. Finally, GNU-201+anti-PD1 resulted in significantly increased levels of Perforin and TNFα producing CD8+ T cells. With GNU-101 there was a trend towards elevated numbers of T cells and TNFα+ CD4 T cells. In mice that received GNU-101 with anti-PD1, we observed significantly elevated levels of TNFα+ CD8+ T cells and a trend towards higher numbers of IFNγ+ CD8+ T cells. GNU-201 and GNU-101 induced a significant modulation of the gut microbiota. In particular, the products promoted the growth of Akkermansia spp., a genus previously associated with anti-tumor immunity. In conclusion, GNU-201 and GNU-101 are both able to boost an anti-tumor immune response upon PD1 inhibition and GNU-101 shows moderate anti-tumor effects on its own. The response is linked with tumor-infiltration and activation of T cells and seems to be associated with changes in the gut microbiome. This study highlights a potential therapeutic role for glycopeptides in boosting anti-tumor immunity in response to ICI therapy. Citation Format: Marianne R. Spalinger, Romain Wyss, Sara Vidal, Yong Miao, Michael Scharl, Yemi Adesokan. Glycopeptides promote anti-cancer immune response against solid tumors [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 5600.

Accurate evaluation of the treatment effects of immunotherapy on subcutaneous ovarian cancer in mice with nonlinear optical imaging and algorithmic analysis.

Immunotherapy and its evaluation have shown great promise for cancer treatment. Here, a mouse subcutaneous transplantable tumor model was applied to testing therapeutic strategies. The mouse model was treated by regulating anti-PD-L1, anti-CTLA-4, cisplatin and their combined therapy. Biochemistry experiments have found that after immunotherapy, mice have more immune responses, which were manifested by an increase in the content of growth factors and the activation of T cells. Meanwhile, multimodal nonlinear optical microscopy imaging combined with algorithms was used to evaluate the treatment's effectiveness. By detecting the metabolism rate and microstructure in tissue, it was proved that combined therapies including immune checkpoint inhibitors do have a better effect on ovarian tumors. Our discovery of valid treatments for mice with ovarian tumor and provides an evaluation tool via nonlinear optics combined with algorithms offers new insights into therapeutic effect.

Role of Extracellular High-Mobility Group Box-1 as a Therapeutic Target of Gastric Cancer.

Background: High-mobility group box-1 (HMGB1) is involved in the tumorigenesis and metastasis of various cancers. The present study investigated the roles of extracellular HMGB1 in the progression of gastric cancer (GC) and the therapeutic effects of recombinant human soluble thrombomodulin (rTM) targeting HMGB1. Methods: The effects of extracellular HMGB1 and rTM on GC cells were assessed using proliferation and Transwell assays. Their effects on local tumor growth and metastasis were evaluated using subcutaneous tumor and liver metastasis mouse models, respectively. Plasma HMGB1 concentrations in GC patients were measured using ELISA. The relationships between plasma HMGB1 concentrations and the prognosis and clinicopathological factors of patients were also investigated. Results: GC proliferation, migration, and invasion abilities were promoted by increases in extracellular HMGB1 concentrations and alleviated by rTM. In the subcutaneous tumor model, local tumor growth was promoted by the addition of rhHMGB1 and alleviated by rTM. Similar changes occurred in the liver metastasis model. Recurrence-free survival (p < 0.01) and overall survival (p = 0.01) were significantly worse in patients with high plasma HMGB1 concentrations. Conclusion: Plasma HMGB1 concentrations are a prognostic marker in GC patients. Extracellular HMGB1 promotes cancer progression and has potential as a novel treatment target in GC cells for rTM.

Cordycepin enhances anti-tumor immunity in colon cancer by inhibiting phagocytosis immune checkpoint CD47 expression

Cordycepin, also known as 3′-deoxyadenosine, is an extract from Cordyceps militaris, which has been reported as an anti-inflammation and anti-tumor substance without toxicity. However, the pharmacological mechanism of Cordycepin on tumor immunity under its anti-tumor effect has not yet been elucidated. Herein, we investigated Cordycepin’s anti-tumor effect on colon cancer both in vitro and in vivo. Our results show that Cordycepin can inhibit growth, migration, and promoted apoptosis of CT26 cells in a dose-dependent manner. Cordycepin suppressed the growth of colon cancer in mouse subcutaneous tumor model by modulating tumor immune microenvironment where CD4+ T, CD8+ T, M1 type macrophages, NK cells were up-regulated. Further investigations revealed that Cordycepin inhibited phagocytosis immune checkpoint CD47 protein expression by reducing BNIP3 expression. In addition, Cordycepin also inhibited the expression of TSP1 in tumor cells and Jurkat cells, which may reduce the binding of TSP1 to CD47, thereby reducing T cell apoptosis and allowing more T cells to infiltrate into tumors. And in vitro co-culture experiments proved that Cordycepin could enhance the phagocytosis of CT26 cells by macrophages. These results explained the underlying mechanism of the anti-tumor immunity of Cordycepin. In conclusion, our results identify a novel mechanism by which Cordycepin inhibits phagocytosis immune checkpoint CD47 in tumor cells to promote tumor cells phagocytosis of macrophages. Cordycepin may be able to serve as a more effective immunotherapeutic drug against colon cancer.

Cascade Drug Delivery through Tumor Barriers of Pancreatic Cancer via Ultrasound in Combination with Functional Microbubbles.

The abundant desmoplastic stroma and the lack of sufficient targets on pancreatic cancer cells render poor drug penetration and cellular uptake, which significantly compromise the chemotherapy efficacy. Herein, we reported a three-step cascade delivery strategy for selective delivery of paclitaxel (PTX) to achieve a targeted therapy for pancreatic cancer. cRGD and cCLT1 peptides, which could target the integrin and fibronectin, respectively, overexpressed in pancreatic cancer cells and stroma, were decorated on PTX-loaded microbubbles, resulting in the formation of dual-targeting PTX-RCMBs. In this strategy, ultrasound in combination with PTX-RCMBs first enhanced the permeability of tumor vessels via cavitation effects and simultaneously helped the generated PTX-RCNPs penetrate into the stroma. Then, the cCLT1 peptide modified on PTX-RCNPs selectively bound the fibronectin highly expressed in the stroma and later targeted the integrin (α5β1) on the cell surface. Finally, another targeting cRGD peptide modified on PTX-RCNPs would further promote PTX uptake via targeting the integrin (αvβ3) on the cell surface. This strategy significantly increased the delivery of PTX into tumor tissues. Moreover, the in vivo effective accumulation of PTX was monitored by ultrasound and fluorescence bimodal imaging. The tumor growth inhibition was investigated on subcutaneous tumor mouse models with 89.8% growth inhibition rate during 21 days of treatment, showing great potential for improving pancreatic cancer therapy.

Heterodimeric RGD-NGR PET Tracer for the Early Detection of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal gastrointestinal cancer, and its poor prognosis is highly associated with the lack of an efficient early detection technology. Here, we report that RGD-NGR heterodimer labeled with PET isotope could be applied in PDAC early detection. The RGD-NGR tracer was first compared with its corresponding monomeric counterparts via PET imaging studies using mice bearing a subcutaneous BxPC3 tumor. Subsequently, the RGD-NGR tracer was evaluated in autochthonous mouse models with spontaneously developed late stage PanIN lesions (KCER mice) or PDAC (KPC mice) via both PET imaging studies and ex vivo biodistribution studies. Furthermore, a comparison between 2-deoxy-2[18F]fluoro-D-glucose ([18F]F-FDG) and the RGD-NGR tracer was conducted via PET imaging of the same KCH mouse bearing spontaneously developed PDAC. H&E staining was performed to confirm the malignant pancreatic tissue in the KCH mouse. Immunofluorescence staining was performed to confirm the expression of integrin αVβ3 and CD13. The RGD-NGR tracer exhibited improved in vivo performance as compared with its corresponding monomeric counterparts on the subcutaneous BxPC3 tumor mouse model. Subsequent evaluation in autochthonous mouse models demonstrated its capability to detect both pre-malignant and malignant pancreases. Further comparison with [18F]F-FDG revealed the superiority of the proposed heterodimer in imaging spontaneously developed PDAC. H&E staining confirmed the malignant pancreatic tissue in the KCH mouse, while the expression of both integrin αVβ3 and CD13 receptors was demonstrated with immunofluorescence staining. The proposed RGD-NGR heterodimer possesses the potential to be applied in the PDAC early detection for high-risk populations.

Cellular membrane-based vesicles displaying a reconstructed B cell maturation antigen for multiple myeloma therapy by dual targeting APRIL and BAFF

Excessive secretion of cytokines (such as APRIL and BAFF) in the bone marrow microenvironment (BMM) plays an essential role in the formation of relapsed or refractory multiple myeloma (MM). Blocking the binding of excessive cytokines to their receptors is becoming a promising approach for MM therapy. Here, we proposed a strategy of engineering cell membrane-based nanovesicles (NVs) to reconstruct B cell maturation antigen (BCMA), a receptor of APRIL and BAFF, to capture excess APRIL/BAFF in BMM as a bait protein. Our results showed that reconstructed BCMA expressed on the membrane of NVs (Re-BCMA-NVs) retained the ability of binding to soluble and surface-bound APRIL/BAFF in BMM. Consequently, Re-BCMA-NVs blocked the activation of the NF-κB pathway, downregulating the expression of anti-apoptosis genes and cell cycle-related genes, and hence inhibiting MM cell survival. Importantly, Re-BCMA-NVs showed a synergistic anti-MM effect when administrated together with bortezomib (BTZ) in vitro and in vivo. Our NVs targeting multiple cytokines in cancer microenvironment provides a solution to enhance sensitivity of MM cells to BTZ-based therapy. Statement of significanceExcessive APRIL and BAFF is reported to promote the survival of MM cell and facilitate the formation of resistance to bortezomib therapy. In this study, we bioengineered cell membrane derived reconstructed BCMA nanovesicles (Re-BCMA-NVs) to capture both soluble and cell-surface APRIL and BAFF. These NVs inhibited the activation of NF-κB pathway and thus inhibit the survival of MM cells in 2D, 3D and subcutaneous mouse tumor models. Importantly, Re-BCMA-NVs showed a synergistic anti-MM effect when administrated together with bortezomib in vitro and in vivo. Taken together, our NVs targeting multiple cytokines in cancer microenvironment provides a solution to enhance sensitivity of MM cells to bortezomib-based therapy.

LncRNA LINC00649 promotes the growth and metastasis of triple-negative breast cancer by maintaining the stability of HIF-1α through the NF90/NF45 complex

ABSTRACT There is no clear treatment guideline or individualized treatment plan for triple-negative breast cancer (TNBC). The aim of this study was to investigate more effective targets for TNBC-targeted therapy. MDA-MB-231 and BT549 cell lines were used to explore the function of LINC00649 on the proliferation, invasion, and migration of TNBC cells. A mice subcutaneous tumor model and a pulmonary metastasis model was established to identify the role of LINC00649 on the growth and metastasis of TNBC in vivo. LINC00649 was found to be a key molecule involved in the occurrence and development of TNBC by screening of public databases and detection of TNBC clinical samples. LINC00649 increased hypoxia-inducible factor 1α (HIF-1α) mRNA stability and protein expression by interacting with the nuclear factor 90 (NF90)/NF45 complex. In vitro, interference with LINC00649 inhibits MDA-MB-231 and BT549 cell proliferation, migration, and invasion, and the addition of HIF-1α revised this effect. In vivo experiments showed that LINC00649 promoted the growth and metastasis of TNBC. We demonstrated that LINC00649 interacts with the NF90/NF45 complex to increase the mRNA stability of HIF-1α and up-regulate HIF-1α expression, thereby inducing the proliferation, invasion, and migration of TNBC cells as well as tumor growth and metastasis.

Phosphatidylserine released from apoptotic cells in tumor induces M2-like macrophage polarization through the PSR-STAT3-JMJD3 axis.

BackgroundUnderstanding how the tumor microenvironment is shaped by various factors is important for the development of new therapeutic strategies. Tumor cells often undergo spontaneous apoptotic cell death in tumor microenvironment, these apoptotic cells are histologically co‐localized with immunosuppressive macrophages. However, the mechanism by which tumor cell apoptosis modulates macrophage polarization is not fully understood. In this study, we aimed to explore the tumor promoting effects of apoptotic tumor cells and the signal pathways involved.MethodsApoptotic cells and macrophages in tumors were detected by immunohistochemical staining. Morphological analysis was performed with Giemsa staining. Lipids generated from apoptotic cells were detected by liquid chromatography‐mass spectrometry. Phosphatidylserine‐containing liposomes were prepared to mimic apoptotic cells. The expression of protein was determined by real‐time PCR, immunohistochemistry enzyme‐linked immunosorbent assay and Western blotting. Mouse malignant ascites and subcutaneous tumor models were designed for in vivo analysis. Transgenic mice with specific genes knocked out and inhibitors specific to certain proteins were used for the mechanistic studies.ResultsThe location and the number of apoptotic cells were correlated with that of macrophages in several types of carcinomas. Phosphatidylserine, a lipid molecule generated in apoptotic cells, induced polarization and accumulation of M2‐like macrophages in vivo and in vitro. Moreover, sustained administration of phosphoserine promoted tumor growth in the malignant ascites and subcutaneous tumor models. Further analyses suggested that phosphoserine induced a M2‐like phenotype in macrophages, which was related to the activation of phosphoserine receptors including T‐cell immunoglobin mucin 4 (TIM4) and the FAK‐SRC‐STAT3 signaling pathway as well as elevated the expression of the histone demethylase Jumonji domain‐containing protein 3 (JMJD3). Administration of specific inhibitors of these pathways could reduce tumor progression.ConclusionsThis study suggest that apoptotic cell‐generated phosphoserine might be a notable signal for immunosuppressive macrophages in tumors, and the related pathways might be potential therapeutic targets for cancer therapy.

Histotripsy Bubble Cloud Contrast With Chirp-Coded Excitation in Preclinical Models.

Histotripsy is a focused ultrasound therapy for tissue ablation via the generation of bubble clouds. These effects can be achieved noninvasively, making sensitive and specific bubble imaging essential for histotripsy guidance. Plane-wave ultrasound imaging can track bubble clouds with an excellent temporal resolution, but there is a significant reduction in echoes when deep-seated organs are targeted. Chirp-coded excitation uses wideband, long-duration imaging pulses to increase signals at depth and promote nonlinear bubble oscillations. In this study, we evaluated histotripsy bubble contrast with chirp-coded excitation in scattering gel phantoms and a subcutaneous mouse tumor model. A range of imaging pulse durations were tested, and compared to a standard plane-wave pulse sequence. Received chirped signals were processed with matched filters to highlight components associated with either fundamental or subharmonic (bubble-specific) frequency bands. The contrast-to-tissue ratio (CTR) was improved in scattering media for subharmonic contrast relative to fundamental contrast (both chirped and standard imaging pulses) with the longest-duration chirped-pulse tested (7.4 [Formula: see text] pulse duration). The CTR was improved for subharmonic contrast relative to fundamental contrast (both chirped and standard imaging pulses) by 4.25 dB ± 1.36 dB in phantoms and 3.84 dB ± 6.42 dB in vivo. No systematic changes were observed in the bubble cloud size or dissolution rate between sequences, indicating image resolution was maintained with the long-duration imaging pulses. Overall, this study demonstrates the feasibility of specific histotripsy bubble cloud visualization with chirp-coded excitation.

Anlotinib Enhances the Antitumor Activity of High-Dose Irradiation Combined with Anti-PD-L1 by Potentiating the Tumor Immune Microenvironment in Murine Lung Cancer.

Background Radioimmunotherapy has become one of the most promising strategies for cancer treatment. Preclinical and clinical studies have demonstrated that antiangiogenic therapy can improve the efficacy of immunotherapy and sensitize radiotherapy through a variety of mechanisms. However, it is undefined whether angiogenesis inhibitors can enhance the effect of radioimmunotherapy. In this study, we aim to explore the role of anlotinib (AL3818) on the combination of radiotherapy and immune checkpoint inhibitors in Lewis lung carcinoma mouse. Methods C57BL/6 mouse subcutaneous tumor model was used to evaluate the ability of different treatment regimens in tumor growth control. Immune response and immunophenotyping including the quantification and activation were determined by flow cytometry, multiplex immunofluorescence, and multiplex immunoassay. Results Triple therapy (radiotherapy combined with anti-PD-L1 and anlotinib) increased tumor-infiltrating lymphocytes and reversed the immunosuppressive effect of radiation on the tumor microenvironment in mouse model. Compared with radioimmunotherapy, the addition of anlotinib also boosted the infiltration of CD8+ T cells and M1 cells and caused a decrease in the number of MDSCs and M2 cells in mice. The levels of IFN-gamma and IL-18 were the highest in the triple therapy group, while the levels of IL-23, IL-13, IL-1 beta, IL-2, IL-6, IL-10, and Arg-1 were significantly reduced. NF-κB, MAPK, and AKT pathways were downregulated in triple therapy compared with radioimmunotherapy. Thus, the tumor immune microenvironment was significantly improved. As a consequence, triple therapy displayed greater benefit in antitumor efficacy. Conclusion Our findings indicate that anlotinib might be a potential synergistic treatment for radioimmunotherapy to achieve better antitumor efficacy in NSCLC patients by potentiating the tumor immune microenvironment.

The Mitochondrial PHB2/OMA1/DELE1 Pathway Cooperates with Endoplasmic Reticulum Stress to Facilitate the Response to Chemotherapeutics in Ovarian Cancer.

Interactions between the mitochondrial inner and outer membranes and between mitochondria and other organelles closely correlates with the sensitivity of ovarian cancer to cisplatin and other chemotherapeutic drugs. However, the underlying mechanism remains unclear. Recently, the mitochondrial protease OMA1, which regulates internal and external signals in mitochondria by cleaving mitochondrial proteins, was shown to be related to tumor progression. Therefore, we evaluated the effect of OMA1 on the response to chemotherapeutics in ovarian cancer cells and the mouse subcutaneous tumor model. We found that OMA1 activation increased ovarian cancer sensitivity to cisplatin in vivo and in vitro. Mechanistically, in ovarian cancer, OMA1 cleaved optic atrophy 1 (OPA1), leading to mitochondrial inner membrane cristae remodeling. Simultaneously, OMA1 induced DELE1 cleavage and its cytoplasmic interaction with EIF2AK1. We also demonstrated that EIF2AK1 cooperated with the ER stress sensor EIF2AK3 to amplify the EIF2S1/ATF4 signal, resulting in the rupture of the mitochondrial outer membrane. Knockdown of OMA1 attenuated these activities and reversed apoptosis. Additionally, we found that OMA1 protease activity was regulated by the prohibitin 2 (PHB2)/stomatin-like protein 2 (STOML2) complex. Collectively, OMA1 coordinates the mitochondrial inner and outer membranes to induce ovarian cancer cell death. Thus, activating OMA1 may be a novel treatment strategy for ovarian cancer.

The combination therapy of Everolimus and anti-PD-1 improves the antitumor effect by regulating CD8+ T cells in bladder cancer.

This study aimed to investigate the efficacy of Everolimus (EVE) in combination with immune checkpoint inhibitors (ICIs) in bladder cancer treatment and the underlying mechanisms. In vitro, MB49 cells were exposed to gradient concentrations (0nM-100nM) of EVE for 48h, to investigate the cell viability and cell proliferative potential. In vivo, we applied a subcutaneous tumor mouse model of bladder cancer and the mice were treated with EVE monotherapy (different doses) or in combination with anti-programmed cell death protein 1 (PD-1) agents to study the impacts on tumor growth and explore the immune mechanism. The influences of treatments on peripheral immune profiles and tumor immune microenvironment were also discussed. EVE could inhibit the growth of MB49 cells in vitro. Though high-dose EVE monotherapy could induce tumor regression in vivo, it also contributed to immunosuppression. High-dose EVE inhibited the expression of PD-L1 by inhibiting Th1 cytokine secretion, while combined therapy with PD-1 inhibitors showed no extra profit. Low-dose EVE in combination with PD-1 inhibitors could effectively suppress tumor growth by increasing periphery CD8+ T cell frequency and GZMB+ CD8+ T cell frequency in the tumor microenvironment. High-dose EVE monotherapy induced tumor regression, but with immunosuppression to some content. Combination therapy with low-dose EVE and PD-1 inhibitor could effectively inhibit the growth of bladder tumors by enhancing the antitumor immunity of CD8+ T cells in both periphery and tumor microenvironment.

MSC-Derived Extracellular Vesicle-Delivered L-PGDS Inhibit Gastric Cancer Progression by Suppressing Cancer Cell Stemness and STAT3 Phosphorylation.

Mesenchymal stem cell- (MSC-) derived extracellular vesicles (EVs) serving as delivery system have attracted extensive research interest, especially in cancer therapy. In our previous study, lipocalin-type prostaglandin D2 synthase (L-PGDS) showed inhibitory effects on gastric cancer growth. In this study, we aimed to explore whether MSC-EV-delivered L-PGDS (EVs-L-PGDS) could inhibit gastric cancer progression. EVs-L-PGDS were generated from MSCs transfected with adenovirus encoding L-PGDS. Cell colony-forming, migration, invasion, and flow cytometry assays were used to show the inhibitory effects of EVs on tumor cells in vitro, and the nude mouse subcutaneous tumor model was performed to show the inhibitory effect of EVs on tumor progression in vivo. In vitro, EVs-L-PGDS could be internalized and inhibit the colony-forming, migration, and invasion ability of gastric cancer cell SGC-7901 and promote cell apoptosis. In vivo, EVs-L-PGDS inhibited the tumor growth in nude mouse subcutaneous tumor-bearing model. Compared with the PBS and EVs containing empty vector (EVs-Vector) group, more apoptotic cells and higher L-PGDS expression were detected in tumor tissue of the EVs-L-PGDS treatment group. And these differences are significant. Mechanistically, EVs-L-PGDS reduced the expression of stem cell markers including Oct4, Nanog, and Sox2 and inhibited STAT3 phosphorylation in gastric cancer cell SGC-7901. In conclusion, our results imply that MSC-derived EVs could be utilized as an effective nanovehicle to deliver L-PGDS for gastric cancer treatment, which provides a novel idea for the EV-based cancer therapy.

Acidity-Activated Charge Conversion of 177Lu-Labeled Nanoagent for the Enhanced Photodynamic Radionuclide Therapy of Cancer.

Nanomaterials in combination with radionuclide therapy (RNT) provide new opportunities for cancer treatment. However, nanomaterials with efficient tumor accumulation have been less exploited for effective radionuclide-based therapy. Here, we report glycol chitosan-based nanoparticles (GCP-NPs) with acidic pH-dependent surface charge conversion for efficient radionuclide-based combination therapy. The nanoplatform can change the surface charge of nanoparticles from slight negative to positive in the acidic tumor microenvironment, which facilitates cellular internalization and penetration and thus improves the tumor accumulation efficiency of nanomaterials. Radiolabeling of GCP-NPs with 99mTc enables in vivo radioactive imaging in the mouse subcutaneous tumor model, showing 8.1-fold enhanced tumor uptake relative to pH-insensitive control nanoparticles (termed as GCOP-NPs). Afterward, therapeutic radioisotope 177Lu-labeled GCP-NPs (177Lu-GCP-NPs) that utilize RNT synergistic with photodynamic therapy (PDT) derived from conjugated pyropheophorbide-a within nanoparticles endow superior antitumor efficacy in living cells and tumor-bearing mouse model. More importantly, the combination of RNT and PDT using 177Lu-GCP-NPs can effectively inhibit lung metastasis and eliminate splenomegaly, which is not possible for individual RNT or PDT. Therefore, this study proposes a facile radionuclide-based combination therapy strategy toward complete cancer remission.