Inoculation Of Cancer Cells Research Articles

Effects of photobiomodulation on colon cancer cell line HT29 according to mitochondria

Background/AimAlthough photobiomodulation therapy (PBMt) is available to alleviate post-operative side effects of malignant diseases, its application is still controversial due to some potential of cancer recurrence and occurrence of a secondary malignancy. We investigated effect of PBMt on mitochondrial function in HT29 colon cancer cells. MethodsHT29 cell proliferation was determined with MTT assay after PBMt. Immunofluorescent staining was performed to determine mitochondrial biogenesis and reactive oxygen species (ROS). Mitochondrial membrane potential was measured with Mitotracker. Western blotting was executed to determine expression of fission, fusion, UCP2, and cyclin B1 and D1 proteins. In vivo study was performed by subcutaneously inoculating cancer cells into nude mice and immunohistochemistry was done to determine expression of FIS1, MFN2, UCP2, and p-AKT. ResultsThe proliferation and migration of HT29 cells reached maximum with PBMt (670 nm, light emitting diode, LED) at 2.0 J/cm2 compared to control (P < 0.05) with more expression of cyclin B1 and cyclin D1 (P < 0.05). Immunofluorescent staining showed that ROS and mitochondrial membrane potential were enhanced after PBMt compared to control. ATP synthesis of mitochondria was also higher in the PBMt group than in the control (P < 0.05). Expression levels of fission and fusion proteins were significantly increased in the PBMt group than in the control (P < 0.05). Electron microscopy revealed that the percentage of mitochondria showing fission was not significantly different between the two groups. Oncometabolites including D-2-hydoxyglutamate in the supernatant of cell culture were higher in the PBMt group than in the control with increased UCP2 expression (P < 0.05). Both tumor size and weight of xenograft in nude mice model were bigger and heavier in the PBMt group than in the control (P < 0.05). Immunohistologically, mitochondrial biogenesis proteins UCP2 and p-AKT in xenograft of nude mice were expressed more in the PBMt group than in the control (P < 0.05). ConclusionsTreatment with PBM using red light LED may induce proliferation and progression of HT29 cancer cells by increasing mitochondrial activity and fission.

Impact of heparanase-2 (Hpa2) on cancer and inflammation: Advances and paradigms.

HPSE2, the gene-encoding heparanase 2 (Hpa2), is mutated in urofacial syndrome (UFS), a rare autosomal recessive congenital disease attributed to peripheral neuropathy. Hpa2 lacks intrinsic heparan sulfate (HS)-degrading activity, the hallmark of heparanase (Hpa1), yet it exhibits a high affinity toward HS, thereby inhibiting Hpa1 enzymatic activity. Hpa2 regulates selected genes that promote normal differentiation, tissue homeostasis, and endoplasmic reticulum (ER) stress, resulting in antitumor, antiangiogenic, and anti-inflammatory effects. Importantly, stress conditions induce the expression of Hpa2, thus establishing a feedback loop, where Hpa2 enhances ER stress which, in turn, induces Hpa2 expression. In most cases, cancer patients who retain high levels of Hpa2 survive longer than patients bearing Hpa2-low tumors. Experimentally, overexpression of Hpa2 attenuates the growth of tumor xenografts, whereas Hpa2 gene silencing results in aggressive tumors. Studies applying conditional Hpa2 knockout (cHpa2-KO) mice revealed an essential involvement of Hpa2 contributed by the host in protecting against cancer and inflammation. This was best reflected by the distorted morphology of the Hpa2-null pancreas, including massive infiltration of immune cells, acinar to adipocyte trans-differentiation, and acinar to ductal metaplasia. Moreover, orthotopic inoculation of pancreatic ductal adenocarcinoma (PDAC) cells into the pancreas of Hpa2-null vs. wild-type mice yielded tumors that were by far more aggressive. Likewise, intravenous inoculation of cancer cells into cHpa2-KO mice resulted in a dramatically increased lung colonization reflecting the involvement of Hpa2 in restricting the formation of a premetastatic niche. Elucidating Hpa2 structure-activity-relationships is expected to support the development of Hpa2-based therapies against cancer and inflammation.

Dynamic Proteomic Changes in Tumor and Immune Organs Reveal Systemic Immune Response to Tumor Development

In orthotopic mouse tumor models, tumor progression is a complex process, involving interactions among tumor cells, host cell-derived stromal cells, and immune cells. Much attention has been focused on the tumor and its tumor microenvironment, while the host’s macroenvironment including immune organs in response to tumorigenesis is poorly understood. Here, we report a temporal proteomic analysis on a subcutaneous tumor and three immune organs (LN, MLN, and spleen) collected on Days 0, 3, 7, 10, 14, and 21 after inoculation of mouse forestomach cancer cells in a syngeneic mouse model. Bioinformatics analysis identified key biological processes during distinct tumor development phases, including an initial acute immune response, the attack by the host immune system, followed by the adaptive immune activation, and the build-up of extracellular matrix. Proteomic changes in LN and spleen largely recapitulated the dynamics of the immune response in the tumor, consistent with an acute defense response on D3, adaptive immune response on D10, and immune evasion by D21. In contrast, the immune response in MLN showed a gradual and sustained activation, suggesting a delayed response from a distal immune organ. Combined analyses of tumors and host immune organs allowed the identification of potential therapeutic targets. A proof-of-concept experiment demonstrated that significant growth reduction can be achieved by dual inhibition of MEK and DDR2. Together, our temporal proteomic dataset of tumors and immune organs provides a useful resource for understanding the interaction between tumors and the immune system and has the potential for identifying new therapeutic targets for cancer treatment.

Establishment of humanised xenograft models as in vivo study for lung metastasis of osteosarcoma.

Humanised xenograft models and cancer cell lines are widely used for preclinical drug evaluation, biological studies, and targeted therapy strategies in cancer research. A humanised mouse model is a laboratory mouse that has been genetically modified to contain specific human genes, cells, or tissues. By introducing human-specific elements into rodents, researchers can create a more accurate representation of human physiological and pathological processes. Lacking an appropriate animal model for osteosarcoma (OS), hindered understanding of underlying mechanisms in OS metastasis progression. Markedly, metastasis influences the prognosis and treatment of osteosarcoma. Gaining insight into the mechanisms and occurrences of metastasis could potentially facilitate oncologists in improving therapies. Hence, it is important to develop a lung metastatic OS model to study the basic biology of its progression. This study has established a tumour-bearing mouse model using HOS-143B cell line which was injected into male NOD.SCID gamma (NSG) mice at two locations; intramuscularly (hind leg) and subcutaneously (back) respectively. The primary and metastatic tumour size was monitored by palpating the area of tumour induced and quantified using digital calliper. H&E staining was performed by pathologist to confirm metastasis. Our results showed that mice injected with 1 million cancer cells were unable to produce tumours. Meanwhile, mice injected with three million cancer cells showed tumour development and lung metastasis after 25 days of cancer cell inoculation. In conclusion, this study has successfully established a lung metastatic OS mouse model that could be useful for biological studies of OS. These findings imply that this model is essential for safety and efficacy before clinical trials, accelerate the translation from basic research to therapeutic applications.

Abstract 2824: Establishing prostate cancer models for orthotopic and bone metastatic growth using the 22Rv1cell line

Abstract Prostate cancer is the second most common cancer in men with new cases diagnosed in 1.4 million men each year. Luckily the 5-year survival rates in developed countries are high reaching over 90%. However, approximately one third will develop a metastatic disease. In metastatic patients, prostate cancer almost always progresses despite the androgen deprivation therapy and is then referred to as “castration-resistant (mCRPC)”. Bone is involved in 80% of patients with metastasis, and the bone lesions are typically osteoblastic. The 22Rv1 human prostate carcinoma cell line is androgen sensitive and shown to express androgen receptor (AR). They also express low levels of prostate-specific membrane antigen (PSMA). The aim of this study was to establish orthotopic and intratibial preclinical in vivo prostate cancer models that can be used in drug development when targeting cancer cells and their local environment. Male athymic nude mice (5-6 weeks old) and NOG mice (over 20 weeks old) were used in the study. 2.5 × 105 22Rv1 cells (ATCC) were inoculated orthotopically into the prostate of NOG mice, and 5 × 105 cells were inoculated intratibially into the bone marrow cavity of athymic nude mice. Tumor growth was followed by PSA measurements every second week using Human PSA ELISA assay. In addition, in the intratibial model, tumor-induced bone changes were monitored by X-ray imaging of the hind limbs. Prostates and hind limbs were collected at sacrifice, fixed in 10% NBF, and processed to paraffin blocks for further histological analysis. Sections of prostate were stained with hematoxylin-eosin (HE) and AR. Tumor-bearing and contralateral (healthy) tibias were stained with HE - OrangeG, MGT, TRAP, and AR. The observed tumor take rate was 92% in the orthotopic and 100% in the intratibial models. Confirmed by IHC, tumors formed in both the orthotopic and intratibial models expressed AR. 22Rv1 cells formed osteoblastic - lytic mixed bone lesions in the intratibial model. Based on lesion areas, randomization of the tumor bearing mice in the intratibial model can be done after 2 weeks from cancer cell inoculation. Taken together, the 22Rv1 prostate cancer models are relatively fast growing, express AR and present good take rates. Furthermore, the intratibial model represents robust tumor-induced changes in bone with strong osteoblastic component. Thus, these models have several advantages in efficacy studies of novel drug candidates for prostate cancer. Citation Format: Justyna Zdrojewska, Katja Fagerlund, Jenni H. Mäki-Jouppila, Mari I. Suominen. Establishing prostate cancer models for orthotopic and bone metastatic growth using the 22Rv1cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2824.

DDEL-12. A CONFORMABLE IMPLANTABLE DEVICE - MICROMESH - FOR THE SUSTAINED DELIVERY OF CHEMOTHERAPEUTIC MOLECULES AND IMMUNE CHECK POINT INHIBITORS AGAINST HIGH-GRADE GLIOMAS

Abstract High-grade gliomas, like glioblastoma (GBM), continue to have a poor prognosis with therapeutic options limited to maximal safe resection and adjuvant radiochemotherapy. Effective treatment is hampered by the blood-brain barrier, limiting the intratumor permeation of systemically administered agents; and biological heterogeneity of malignant cells, requiring complex therapeutic regimens. A conformable, compartmentalized polymeric implant – microMESH – has been engineered to overcome these obstacles by realizing a localized, sustained release of drugs, small inhibitors, antibodies, and nanomedicines. In the present configuration, microMESH comprises a water-soluble polyvinyl alcohol microlayer, loaded with the immune checkpoint aCD47, intercalated with a microfabricated network of poly lactic-co-glycolic acid, carrying docetaxel (DTXL). Upon deposition over the tumor margins, microMESH adheres and conforms to the local tissue releasing thereof its therapeutic cargo for several weeks, circumventing possible systemic toxicity. Gel permeation chromatography, nuclear magnetic resonance spectroscopy, weight and pH variations were measured to assess the microMESH biodegradation over time. microMESH biocompatibility and cytotoxicity were assessed on cancer cell monolayers and spheroids. The preclinical performance of microMESH was evaluated in two syngeneic mouse models of high-grade gliomas obtained via the intracranial inoculation of murine cancer cells – CT-2A, cold tumor; GL261, hot tumor. Flow cytometry was used to map the immune component in the tumor and assess the expression of immune checkpoints. Bioluminescence imaging was used to monitor tumor proliferation longitudinally. microMESH biodegraded in ~2 months with no documented toxicity. Preliminary experiments with an aCD47/DTXL–microMESH applied over the unresected margins of an orthotopic CT-2A model documented a median overall survival of 33.5 days against 18 days for untreated mice, 22 days for intravenously injected DTXL, 24 days for intraperitoneally administered aCD47 (statistically not significant difference among the control groups). microMESH allowed the sustained delivery of complex combination therapies directly at the tumor bed demonstrating superior efficacy over systemic therapies.

The Effects of Glypican-3 Deficiency on Radiosensitivity in Liver Cancer Cells

Glypican-3 (GPC-3), a heparan sulfate proteoglycan involved in cellular proliferation, modulates signaling of FGF/FGFR, IGF/IGFR, HGF/Met, Wnt/Frizzled, among others and correlates with survival. GPC-3 is overexpressed in the majority of hepatocellular carcinoma and hepatoblastoma, but not in normal hepatocytes. Accordingly, it is being investigated as a liver cancer-selective target for radiopharmaceutical imaging and therapy. However, the potential linkage between GPC-3 expression and radiosensitivity has not yet been defined. In this study, we investigated the effects of GPC-3 deficiency on radiosensitivity in liver cancer cell lines. CRISPR/Cas9 system was used to engineer GPC-3 knockout variants of liver cancer cell lines, HepG2 & Hep3B, both of which natively express GPC-3. Confirmation of knockout of GPC-3 was evaluated by RT-PCR, western blotting, flow cytometry, immunocytochemistry, and gDNA sequencing. Cell growth and migration were evaluated by BrdU insertion and wound-healing assays, respectively. In vitro radiosensitivity was examined by radiation-induced apoptosis/necrosis (Annexin V-APC and PI staining), cell cycle modification, γH2AX foci formation, and clonogenic assays (6 Gy). Wildtype and knockout lines were engrafted into athymic mice to assess tumor growth kinetics. RT-PCR, western blotting, flow cytometry, and immunocytochemistry all confirmed GPC-3 knockout in both HepG2 and Hep3B cell lines. Nucleotide deletion at exon 3 of the GPC-3 gene was confirmed by gDNA sequencing in HepG2ΔGPC3 and Hep3BΔGPC3. GPC-3 deficiency reduced liver cancer cell proliferation (HepG2ΔGPC3, p = 0.027, and Hep3BΔGPC3, p = 0.031) and migration (HepG2ΔGPC3: 1.5-fold, p<0.001, and Hep3BΔGPC3: 2.3-fold, p<0.001) significantly when compared with wild type. GPC-3 deficiency reduced cell survival and clonogenicity (HepG2ΔGPC3: DEF = 1.23, Hep3BΔGPC3: DEF = 1.23) in liver cancer cells exposed to irradiation (6 Gy). The delayed repair of double-stranded DNA damage was observed in irradiated GPC-3 deficient liver cancer cells. Tumor growth was dramatically delayed by GPC-3 deficiency. Tumor weight measured at 50 (Hep3B) and 60 (HepG2) days after liver cancer cell inoculation corroborated these effects. Knockout lines of HepG2 and Hep3B exhibited decreased cell proliferation, migration, and in vivo tumor growth compared to wildtype. GPC-3 deficiency was associated with increased sensitivity to radiation therapy. Studies identifying the pathways through which this radiosensitivity is mediated are ongoing.

Moxibustion with seed-size moxa cone enhances anti-tumor effect of cyclophosphamide on Hepa1-6 hepatoma-bearing mice by regulating apoptosis factor cysteine family

To observe the anti-tumor effect of moxibustion with seed-sized moxa cones on Hepa1-6 liver cancer bearing (HLCB) mice and its regulatory mechanism on cell apoptosis. A total of 40 male C57BL/6 mice were randomly divided into control, moxibustion, cyclophosphamide (CTX) and moxibustion+CTX groups, with 10 mice in each group. The HLCB model was established by subcutaneous inoculation of Hepa1-6 cancer cells into the right armpit. Mice of the CTX and moxibustion+CTX groups were given intraperitoneal injection of CTX (30 mg/kg), once daily for 3 days. Moxibustion with seed-sized moxa cones were applied to "Dazhui" (GV14), bilateral"Zusanli" (ST36) and "Sanyinjiao" (SP6), with 5 moxa cones for each acupoint, once daily for 10 consecutive days. The survival status scores and body weight of HLCB mice were observed, and the tumor weight and tumor inhibition rate were detected. HE staining was used to observe the morphological changes of tumor tissue. ELISA was used to detect the levels of serum interleukin (IL)-2, IL-4 and tumor necrosis factor-α (TNF-α). Western blot and fluorescent quantitative real-time PCR were used to detect the protein and mRNA expressions of cysteine aspartate protease (Caspase) -3 and Caspase-9 in tumor tissues, separately. Compared with the control group, the survival status scores, body weight, serum IL-2 and TNF-α levels were significantly increased (P<0.05, P<0.01), the tumor weight and serum IL-4 levels were significantly decreased (P<0.05) in the moxibustion group；while the survival status, body weight, tumor weight, serum IL-2 and IL-4 levels were significantly decreased (P<0.01, P<0.05), the content of TNF-α was significantly increased (P<0.01) in the CTX group. The protein and mRNA expressions of Caspase-3 and Caspase-9 in the 3 trentment groups were significantly increased (P<0.05, P<0.01). In comparison with the moxibustion group, the survival status scores, body weight and tumor weight, serum content of IL-2 were significantly decreased (P<0.01, P<0.05). In contrast to the CTX group, the survival status scores, body weight, serum IL-2 and TNF-α content, and the expressions of Caspase-3 and Caspase-9 in tumor tissue were significantly increased (P<0.01, P<0.05), and the tumor weight and serum IL-4 content were significantly decreased (P<0.05) in the moxibustion+CTX group. Results of HE staining showed that the tumor cells in the control group had clear nuclear membranes and nucleoli, with more dividing cells； while less nuclear division and an increase in tumor necrosis areas were found in the 3 treatment groups. Moxibustion with seed-size moxa cone can enhance the anti-tumor effect of CTX and improve the quality of life of HLCB mice, which may be related with its effect in activating the expressions of Caspase-3 and Caspase-9 in tumor tissue.

Novel strategy of liver cancer treatment with modified antisense oligonucleotides targeting human vasohibin-2.

Vasohihibin-2 (VASH2) is a homolog of vasohibin-1 (VASH1) and is overexpressed in various cancers. Vasohihibin-2 acts on both cancer cells and cancer microenvironmental cells. Previous analyses have shown that VASH2 promotes cancer progression and abrogation of VASH2 results in significant anticancer effects. We therefore propose VASH2 to be a practical molecular target for cancer treatment. Modifications of antisense oligonucleotide (ASO) such as bridged nucleic acids (BNA)-based modification increases the specificity and stability of ASO, and are now applied to the development of a number of oligonucleotide-based drugs. Here we designed human VASH2-ASOs, selected an optimal one, and developed 2',4'-BNA-based VASH2-ASO. When systemically administered, naked 2',4'-BNA-based VASH2-ASO accumulated in the liver and showed its gene-silencing activity. We then examined the effect of 2',4'-BNA-based VASH2-ASO in liver cancers. Intraperitoneal injection of naked 2',4'-BNA-based VASH2-ASO exerted a potent antitumor effect on orthotopically inoculated human hepatocellular carcinoma cells. The same manipulation also showed potent antitumor activity on the splenic inoculation of human colon cancer cells for liver metastasis. These results provide a novel strategy for the treatment of primary as well as metastatic liver cancers by using modified ASOs targeting VASH2.

Therapeutic effect and mechanism of Xiaoyao Kangai Jieyu Recipe on mice with breast cancer related depression through regulating COX pathway

This study aimed to investigate the intervention effect and mechanism of Xiaoyao Kangai Jieyu Recipe(XKJR) on hip-pocampal microglia and neuronal damage in mice with breast cancer related depression. The mouse model of breast cancer related depression was established by inoculation of 4T1 breast cancer cells in axilla and subcutaneous injection of corticosterone(30 mg·kg~(-1)). The successfully modeled mice were randomly divided into a model group, a positive drug group(capecitabine 60 mg·kg~(-1)+fluoxetine 19.5 mg·kg~(-1)), and XKJR group(19.5 mg·kg~(-1) crude drug), with 6 in each group. Another 6 normal mice were taken as a normal group. The administration groups were given corresponding drugs by gavage, while the normal and model groups were given an equal volume of distilled water, once a day for 21 consecutive days. The depressive behavior of mice was assessed by glucose consumption test, open field test and novelty-suppressed feeding test. Hematoxylin and eosin(HE) staining and tumor suppression rate were used to evaluate the changes of axillary tumors. The mRNA expressions and the relative protein expressions of interleukin-1β(IL-1β), interleukin-18(IL-18), cyclooxyganese-2(COX-2) and glutamyl-prolyl-tRNA synthetase(EPRs) in the hippocampus of mice were determined by quantitative real-time polymerase chain reaction(qRT-PCR) and immunohistochemistry, respectively. Immunofluorescence was performed to detect the mean fluorescence intensity of CD11b, a marker of hippocampal microglia activation. Nissler staining and transmission electron microscopy were employed to observe the morphological changes and the ultramorphological changes of hippocampal neurons, respectively. The experimental results indicated that compared with the normal group, the model group had reduced glucose consumption and lowered number of total activities in open field test(P&lt;0.05, P&lt;0.01), prolonged first feeding latency in no-velty-suppressed feeding test(P&lt;0.01), and significant depression-like behavior; the contents of IL-1β, IL-18, COX-2, and EPRs in hippocampus were increased(P&lt;0.05, P&lt;0.01), with hippocampal microglia activation and obvious neuronal damage. Compared with the model group, the positive drug group and the XKJR group presented an improvement in depressive behaviors, a decrease in the contents of IL-1β, IL-18, COX-2 and EPRs in hippocampus, and an alleviation in the activation of hippocampal microglia and neuronal damage; the tumor suppression rates of positive drug and XKJR were 40.32% and 48.83%, respectively, suggesting a lower tumor growth rate than that of the model group. In summary, XKJR may improve hippocampal microglia activation and neuronal damage in mice with breast cancer related depression through activating COX signaling pathway.

Relationship between EGFR inhibition in lung adenocarcinoma, cell surface expression of the placental antigen ALPP, and efficacy of ALPP-ADC therapy.

e15018 Background: In normal tissues placental alkaline phosphatases (ALPP) and ALPPL2 are exclusively expressed in the placenta. Their discovery in the cancer surfaceome has opened the possibility of targeting ALPP/ALPPL2. However, their low expression in most cancer types limits their therapeutic potential. In this study, we report a novel mechanism of ALPP upregulation in cancer cells that renders these cells more susceptible to ALPP-targeting therapies, which we evidence in the context of lung adenocarcinoma. Methods: Surface expression of ALPP and ALPPL2 was determined following biotinylation tagging of the cancer surfaceome, isolation of biotinylated proteins using monomeric avidin and mass spectrometry of the biotinylated trypsin digested fraction. ALPP antibody was conjugated with Monomethylauristatin F (ALPP-ADC-MMAF). For orthotopic xenograft models of LUAD were injected. For gefitinib treatment, Mice xenotransplanted with H1650 were treated with gefitinib (50mg/kg) daily for 10 days starting from Day 17 post cancer cell inoculation. For the gefitinib/ALPP-ADC combinational treatment, mice received anti-ALPP-MMAF (5mg/kg) at Day 19 and Day 26. Results: We assessed ALPP and ALPPL2 surfaceome protein levels in 158 cancer cell lines and their mRNA expression levels in 10,967 tumors representing 32 cancer types from The Cancer Genome Atlas (TCGA), which revealed ALPP, and to a lesser extent ALPPL2, to be variably expressed in several cancer types including lung adenocarcinoma (LUAD). We further confirmed surface expression of ALPP by tissue microarray analysis of 204 lung tumors. We report that treatment of LUAD cells with EGFR inhibitors or induction of cancer cell quiescence greatly enhanced ALPP surface expression. Mechanistically, repression of EGFR signaling resulted in activation of the transcription factor FoxO3a, which triggered expression of ALPP. Using xenograft models of LUAD, we further demonstrate that gefitinib treatment significantly upregulated surface expression of ALPP in LUAD tumors but not normal tissue. Combination therapy with gefitinib and an ALPP-ADC-MMAF resulted in superior anti-cancer efficacy compared with gefitinib or ALPP-ADC-MMAF alone. Conclusions: Our study provides mechanistic insights into ALPP upregulation in cancer cells and establishes FoxO3a as a transcriptional regulator of ALPP. Importantly, our findings have yielded a novel ‘two-hit’ combinational therapeutic strategy with enhanced ALPP targeting for greater efficacy.

Abstract 2578: Phosphorylation of AHR by PLK1 promotes metastasis of LUAD via upregulation of DIO2

Abstract Non-small cell lung cancer (NSCLC) is one of the most aggressive cancer types, and metastasis of cancer cells is the major reason leading to death of patients. Aryl hydrocarbon receptor (AHR) is an important transcription factor involved in the initiation and progression of lung cancer. Polo-like kinase 1 (PLK1), a serine/threonine kinase, is another oncogene that promotes the malignancy of multiple cancer types. Preliminary examination indicates that AHR is a potential substrate of PLK1. Nonetheless, the phosphorylation and interaction of these two factors, as well as the subsequent biological significance in lung cancer remain to be determined. Here we prove that PLK1 phosphorylates AHR at serine 489 (S489) in lung adenocarcinoma (LUAD), a subtype of NSCLC. Overexpressing of exogenous AHR with mutation of phosphomemetic amino acid (S489D), enhances migration and invasion in transwell assays compared to cells harboring AHR that is unable to be phosphorylated (S489A), indicating that phosphorylation of AHR by PLK1 induces the metastatic potential of lung cancer. This is further confirmed in mouse studies using intravenous or subcutaneous inoculation of cancer cells. RNA-seq analyses of cells with S489D or S489A show that type 2 deiodinase (DIO2) is underrepresented in S489A but overexpressed in S489D. DIO2 converts thyroxine (T4) to triiodothyronine (T3), which is the more active form of thyroid hormone and activates downstream effect. Treatment with T3 or T4 promotes the metastasis of LUAD, whereas depletion of DIO2 or treatment with deiodinase inhibitor iopanoic acid disrupts this property. Furthermore, this phenomenon is verified by in vivo mouse experiment using intravenous injection of DIO2-depleted cancer cells. Taken together, these results identify phosphorylation of AHR by PLK1 as a mechanism that leads to progression of LUAD. Citation Format: Chaohao Li, Daheng He, Fengyi Mao, Xinyi Wang, Yanning Hao, Yifan Kong, Christine F. Brainson, Jinghui Liu, Yanquan Zhang, Ruixin Wang, Qiongsi Zhang, Zhiguo Li, Xiongjian Rao, Sai Wu, Chi Wang, Qiou Wei, Jianlin Wang, Xiaoqi Liu. Phosphorylation of AHR by PLK1 promotes metastasis of LUAD via upregulation of DIO2 [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 2578.

Schwann cell insulin-like growth factor receptor type-1 mediates metastatic bone cancer pain in mice

Insulin growth factor-1 (IGF-1), an osteoclast-dependent osteolysis biomarker, contributes to metastatic bone cancer pain (MBCP), but the underlying mechanism is poorly understood. In mice, the femur metastasis caused by intramammary inoculation of breast cancer cells resulted in IGF-1 increase in femur and sciatic nerve, and IGF-1-dependent stimulus/non-stimulus-evoked pain-like behaviors. Adeno-associated virus-based shRNA selective silencing of IGF-1 receptor (IGF-1R) in Schwann cells, but not in dorsal root ganglion (DRG) neurons, attenuated pain-like behaviors. Intraplantar IGF-1 evoked acute nociception and mechanical/cold allodynia, which were reduced by selective IGF-1R silencing in DRG neurons and Schwann cells, respectively. Schwann cell IGF-1R signaling promoted an endothelial nitric oxide synthase-mediated transient receptor potential ankyrin 1 (TRPA1) activation and release of reactive oxygen species that, via macrophage-colony stimulating factor-dependent endoneurial macrophage expansion, sustained pain-like behaviors. Osteoclast derived IGF-1 initiates a Schwann cell-dependent neuroinflammatory response that sustains a proalgesic pathway that provides new options for MBCP treatment.

Therapeutic Performance Evaluation of 213Bi-Labelled Aminopeptidase N (APN/CD13)-Affine NGR-Motif ([213Bi]Bi-DOTAGA-cKNGRE) in Experimental Tumour Model: A Treasured Tailor for Oncology

Since NGR-tripeptides (asparagine-glycine-arginine) selectively target neoangiogenesis-associated Aminopeptidase N (APN/CD13) on cancer cells, we aimed to evaluate the in vivo tumour targeting capability of radiolabelled, NGR-containing, ANP/CD13-selective [213Bi]Bi-DOTAGA-cKNGRE in CD13pos. HT1080 fibrosarcoma-bearing severe combined immunodeficient CB17 mice. 10 ± 1 days after cancer cell inoculation, positron emission tomography (PET) was performed applying [68Ga]Ga-DOTAGA-cKNGRE for tumour verification. On the 7th, 8th, 10th and 12th days the treated group of tumourous mice were intraperitoneally administered with 4.68 ± 0.10 MBq [213Bi]Bi-DOTAGA-cKNGRE, while the untreated tumour-bearing animals received 150 μL saline solution. In addition to body weight (BW) and tumour volume measurements, ex vivo biodistribution studies were conducted 30 and 90 min postinjection (pi.). The following quantitative standardised uptake values (SUV) confirmed the detectability of the HT1080 tumours: SUVmean and SUVmax: 0.37 ± 0.09 and 0.86 ± 0.14, respectively. Although no significant difference (p ≤ 0.05) was encountered between the BW of the treated and untreated mice, their tumour volumes measured on the 9th, 10th and 12th days differed significantly (p ≤ 0.01). Relatively higher [213Bi]Bi-DOTAGA-cKNGRE accumulation of the HT1080 neoplasms (%ID/g: 0.80 ± 0.16) compared with the other organs at 90 min time point yields better tumour-to-background ratios. Therefore, the therapeutic application of APN/CD13-affine [213Bi]Bi-DOTAGA- cKNGRE seems to be promising in receptor-positive fibrosarcoma treatment.

Antitumor Effects of Deep Ultraviolet Irradiation for Pancreatic Cancer.

Deep ultraviolet (DUV) light spans within the 250 nm to 350 nm invisible wavelength range. Although it strongly damages various cells, the efficacy of DUV irradiation on pancreatic cancer cells has never been clarified. The purpose of this study was to reveal the antitumor effects of DUV irradiation on pancreatic cancer cells. Human pancreatic cancer cell lines were eradicated with DUV or ultraviolet A (UVA) for 5 s. Several angiogenesis-related proteins were studied in cancer cells after DUV irradiation using a protein antibody array. A subcutaneous xenograft model was established by inoculation of pancreatic cancer cells into mice. Tumors in this model were irradiated with DUV or UVA once or twice for two weeks. Tumor volumes in these groups (DUV×1: one irradiation, DUV×2: two irradiations, and untreated) were analyzed one week after the second irradiation. DUV irradiation significantly changed the cytomorphology of pancreatic cancer cells. In addition, DUV irradiation induced apoptosis on pancreatic cancer cells more strongly than UVA irradiation and no irradiation. Interestingly, lower expression of thrombospondin 1 (TSP1) and tissue inhibitor of metalloproteinase 1 (TIMP1) was identified after DUV treatment. The tumor volume in the DUV-treated groups (DUV×1 and DUV×2) was smaller than that in the untreated group. Further investigations are required to reveal the precise mechanisms of the antitumor effects of DUV irradiation and to facilitate its clinical application as a new therapy for pancreatic cancer. Overall, DUV irradiation can be potentially used as a therapeutic option of pancreatic malignancy.

PD-1 Blockade Combined with Heated Intraperitoneal Chemotherapy Improves Outcome in Experimental Peritoneal Metastases from Colonic Origin in a Murine Model.

Heated intraperitoneal chemotherapy (HIPEC) was shown to induce immunogenicity of peritoneal metastases from colorectal cancer (PM-CRC) by induction of immunogenic cell death. We aimed to explore whether the addition of a checkpoint inhibitor would augment the effect of HIPEC in an experimental murine model of PM-CRC. PM-CRC was established in C57BL mice by intraperitoneal inoculation of MC38 colon cancer cells. HIPEC was administered using the closed technique with mitomycin C (MMC). Clinical and immunological parameters were compared between animals treated with HIPEC alone and those treated with HIPEC+anti-programmed death receptor-1 (aPD-1). MMC-based HIPEC increased the overall survival of animals compared with sham-treated animals (22.8; 95% confidence interval [CI] 21.14-24.53 vs. 18.9 days; 95% CI 17.6-20.3, p<0.001). The extent of peritoneal disease as measured by the modified peritoneal carcinomatosis index was also reduced by HIPEC. This clinical benefit was accompanied by increased infiltration of CD8+, CD68+, and CD20+ cells into tumor metastases in HIPEC-treated animals compared with sham-treated animals. We identified heat shock protein (HSP)90 as a potential immunogenic cell death protein whose expression is increased under HIPEC conditions (fold change: 2.37±1.5 vs. 1 without HIPEC, p<0.05). Combined HIPEC+PD-1 treatment ameliorated survival compared with HIPEC alone and sham treatment (24.66; 95% CI 20.13-29.2 vs. 19; 95% CI 15.85-22.14 and 14.33 days; 95% CI 9.6-19.04, respectively; p=0.008). This clinical effect was accompanied by increased CD8+ tumor infiltration. HIPEC induced the expression of immunogenic cell death signals that can support an anti-tumor immune response. This response can be further exploited by a checkpoint inhibitor.

Interleukin-6 initiates muscle- and adipose tissue wasting in a novel C57BL/6 model of cancer-associated cachexia.

Cancer-associated cachexia (CAC) is a wasting syndrome drastically reducing efficacy of chemotherapy and life expectancy of patients. CAC affects up to 80% of cancer patients, yet the mechanisms underlying the disease are not well understood and no approved disease-specific medication exists. As a multiorgan disorder, CAC can only be studied on an organismal level. To cover the diverse aetiologies of CAC, researchers rely on the availability of a multifaceted pool of cancer models with varying degrees of cachexia symptoms. So far, no tumour model syngeneic to C57BL/6 mice exists that allows direct comparison between cachexigenic- and non-cachexigenic tumours. MCA207 and CHX207 fibrosarcoma cells were intramuscularly implanted into male or female, 10-11-week-old C57BL/6J mice. Tumour tissues were subjected to magnetic resonance imaging, immunohistochemical-, and transcriptomic analysis. Mice were analysed for tumour growth, body weight and -composition, food- and water intake, locomotor activity, O2 consumption, CO2 production, circulating blood cells, metabolites, and tumourkines. Mice were sacrificed with same tumour weights in all groups. Adipose tissues were examined using high-resolution respirometry, lipolysis measurements in vitro and ex vivo, and radioactive tracer studies in vivo. Gene expression was determined in adipose- and muscle tissues by quantitative PCR and Western blotting analyses. Muscles and cultured myotubes were analysed histologically and by immunofluorescence microscopy for myofibre cross sectional area and myofibre diameter, respectively. Interleukin-6 (Il-6) was deleted from cancer cells using CRISPR/Cas9 mediated gene editing. CHX207, but not MCA207-tumour-bearing mice exhibited major clinical features of CAC, including systemic inflammation, increased plasma IL-6 concentrations (190pg/mL, P≤0.0001), increased energy expenditure (+28%, P≤0.01), adipose tissue loss (-47%, P≤0.0001), skeletal muscle wasting (-18%, P≤0.001), and body weight reduction (-13%, P≤0.01) 13days after cancer cell inoculation. Adipose tissue loss resulted from reduced lipid uptake and -synthesis combined with increased lipolysis but was not associated with elevated beta-adrenergic signalling or adipose tissue browning. Muscle atrophy was evident by reduced myofibre cross sectional area (-21.8%, P≤0.001), increased catabolic- and reduced anabolic signalling. Deletion of IL-6 from CHX207 cancer cells completely protected CHX207IL6KO -tumour-bearing mice from CAC. In this study, we present CHX207 fibrosarcoma cells as a novel tool to investigate the mediators and metabolic consequences of CAC in C57BL/6 mice in comparison to non-cachectic MCA207-tumour-bearing mice. IL-6 represents an essential trigger for CAC development in CHX207-tumour-bearing mice.

Human amniotic epithelial cells exert anti-cancer effects through secretion of immunomodulatory small extracellular vesicles (sEV)

We identified here mechanism by which hAECs exert their anti-cancer effects. We showed that vaccination with live hAEC conferred effective protection against murine colon cancer and melanoma but not against breast cancer in an orthotopic cancer cell inoculation model. hAEC induced strong cross-reactive antibody response to CT26 cells, but not against B16F10 and 4T1 cells. Neither heterotopic injection of tumor cells in AEC-vaccinated mice nor vaccination with hAEC lysate conferred protection against melanoma or colon cancer. Nano-sized AEC-derived small-extracellular vesicles (sEV) (AD-sEV) induced apoptosis in CT26 cells and inhibited their proliferation. Co-administration of AD-sEV with tumor cells substantially inhibited tumor development and increased CTL responses in vaccinated mice. AD-sEV triggered the Warburg’s effect leading to Arginine consumption and cancer cell apoptosis. Our results clearly showed that it is AD-sEV but not the cross-reactive immune responses against tumor cells that mediate inhibitory effects of hAEC on cancer development. Our results highlight the potential anti-cancer effects of extracellular vesicles derived from hAEC.

Abstract 592: TLT2 expressed on B cells and Bregs suppresses T cell activity, and treatment of the anti-TLT2 antibody, GENA-105S05, induces tumor growth inhibition in a syngeneic mouse model

Abstract TLT2 (TREML2, Triggering Receptor Expressed on Myeloid cells Like transcript 2) is a single-pass type I membrane protein that contains an immunoglobulin-like domain. Previous studies have shown that TLT2 is up-regulated in response to inflammation in macrophages, suggesting a potential role in the pro-inflammatory response. However, the function of TLT2 in immune cells and crosstalk of cells expressing TLT2 with other immune cells, e.g., T cells, have not been studied. Therefore, we tried to search immune cells expressing TLT2 and explore the effect of TLT2 on T cell activity. T cell proliferation and cytokine secretion assays using TLT2 recombinant protein showed that TLT2 significantly inhibits T cells proliferation and cytokine secretion (IFN gamma and TNF alpha) more than PD-L1. Since TLT2 can act as an immune checkpoint protein, FACS analysis was performed on various cancer cell lines and immune cells to confirm the expression of TLT2. TLT2 was commonly expressed on human and mouse B cells but not cancer cells. Additionally, we found that TLT2 was expressed on human and mouse regulatory B cells (Bregs). Bregs represent a subpopulation of B cells that inhibit effector T cells but induce regulatory T cells and attenuate the anti-tumor effect of tumor-infiltrating immune cells in the tumor microenvironment. Bregs are also closely related to various clinicopathological factors and the survival rates of cancer patients. In particular, Bregs are observed to be high in the patient group not responding to the administration of immunotherapy. Therefore, Bregs could be a potential therapeutic target for immunotherapy of cancer patients. For in vivo function study of TLT2, we inoculated cancer cells in TLT2 WT (wild-type) and KO (knockout) mice and monitored the tumor growth. The size of tumors inoculated in TLT2 KO mice was reduced compared to TLT2 WT mice. In addition, the total population of CD4+ T cells and the population of CD4+ and CD8+ T cells that produce cytokines (IFN gamma and TNF alpha) were increased in the spleen and tumor-infiltrated lymphocyte of the TLT2 KO mice. Finally, we investigated the potential of TLT2 as a cancer immunotherapeutic target using a GENA-105S05, a monoclonal antibody against mouse Tlt2. GENA-105S05 significantly inhibited tumor growth in the MC38 syngeneic mouse tumor model. Further investigation on the function of TLT2 expressed in the Bregs and the development of a monoclonal antibody targeting TLT2 could lead to novel therapeutic interventions. Citation Format: Mi Young Cha, Bu-Nam Jeon, Hyunsuk Yang, Seungmin Byun, Areum Jeong, Joo-Yeon Chung, Sanggyun Kim, Kyung Mi Park, Hansoo Park. TLT2 expressed on B cells and Bregs suppresses T cell activity, and treatment of the anti-TLT2 antibody, GENA-105S05, induces tumor growth inhibition in a syngeneic mouse model [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 592.

Abstract 2093: PEGylated IL-2 with enhanced anti-tumor efficacy and safety profile

Abstract Introduction: Interleukin-2 (IL-2) can activate and expand tumor killing lymphocytes. High doses of IL-2 are needed to activate CD8+ T cells. Severe toxic side effects associated with high doses, as well as the poor PK profile of IL-2, have limited its clinical usage. Various strategies have been employed to prolong half-lives of IL-2 and enable selectivity or bias towards receptor binding to reduce toxicity and maintain good efficacy. Among these, certain PEGylation strategies have generated promising clinical results, but also showed some major limitations. We report a unique PEGylation strategy to produce PEGylated IL-2 with a therapeutic window more than two times larger than the reference PEGylated IL-2. Methods: CT26 murine syngeneic model was used for evaluation of in vivo efficacy and safety. Each 6-8 weeks old female BALB/c mouse was inoculated subcutaneously in the right front flank region with CT26 tumor cells (5 x 105). Tumors were allowed to grow to mean size ~110 mm3 before randomization and treatment. Body weights and tumor volumes were measured three times per week. The tumor-free mice at the end of the study were re-challenged with CT26 colon tumor cells on the left side. Results: Utilizing our proprietary PEGylation technology, rhIL-2 was covalently connected with multiple PEG polymers through releasable linkers (RL). The releasable linkers are designed to modulate the release rate of the active IL-2 species. In CT26 syngeneic mouse model, CS-PEG-IL-2 showed significantly enhanced efficacy and safety over IL-2 and reference PEGylated IL-2. rhIL-2 (2 mg/kg, bid x 5, two cycles, i.p.) resulted in complete response (CR) in one out of eight (1/8) mice, while 3 mg/kg dose resulted in death in 8/8 mice. With the reference PEGylated IL-2, 3/8 CR were obtained at 3 mg/kg dose (q1w x 4, i.v.), and 6 mg/kg dose resulted in death in 8/8 mice. Whereas, with three CS PEGylated IL-2 analogs, 5/8 and 6/8 CR were achieved at 3 mg/kg and 6 mg/kg dose respectively, and no obvious toxicity was observed even at the highest dose. After re-challenged with another inoculation of CT26 cancer cells, the CR mice remained tumor-free until the end of the study at day 158. Conclusions: CS unique PEGylation strategy generated PEG-IL-2 with superior anti-tumor efficacy and safety profiles. Preclinical results support clinical evaluation of this novel class of PEGylated IL-2 analogs. Citation Format: Yuntao Song, Hui Li, Wei Zhang, Dayan Fu, Meng Xu. PEGylated IL-2 with enhanced anti-tumor efficacy and safety profile [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 2093.