Tumor Allograft Research Articles

Overcoming Irinotecan Resistance by Targeting Its Downstream Signaling Pathways in Colon Cancer

Among the most popular chemotherapeutic agents, irinotecan, regarded as a prodrug belonging to the camptothecin family that inhibits topoisomerase I, is widely used to treat metastatic colorectal cancer (CRC). Although immunotherapy is promising for several cancer types, only microsatellite-instable (~7%) and not microsatellite-stable CRCs are responsive to it. Therefore, it is important to investigate the mechanism of irinotecan function to identify cellular proteins and/or pathways that could be targeted for combination therapy. Here, we have determined the effect of irinotecan treatment on the expression/activation of tumor suppressor genes (including p15Ink4b, p21Cip1, p27Kip1, and p53) and oncogenes (including OPN, IL8, PD-L1, NF-κB, ISG15, Cyclin D1, and c-Myc) using qRT-PCR, Western blotting, immunofluorescence (IF), and RNA sequencing of tumor specimens. We employed stable knockdown, neutralizing antibodies (Abs), and inhibitors of OPN, p53, and NF-κB to establish downstream signaling and sensitivity/resistance to the cytotoxic activities of irinotecan. Suppression of secretory OPN and NF-κB sensitized colon cancer cells to irinotecan. p53 inhibition or knockdown was not sufficient to block or potentiate SN38-regulated signaling, suggesting p53-independent effects. Irinotecan treatment inhibited tumor growth in syngeneic mice. Analyses of allograft tumors from irinotecan-treated mice validated the cell culture results. RNA-seq data suggested that irinotecan-mediated activation of NF-κB signaling modulated immune and inflammatory genes in mice, which may compromise drug efficacy and promote resistance. In sum, these results suggest that, for CRCs, targeting OPN, NF-κB, PD-L1, and/or ISG15 signaling may provide a potential strategy to overcome resistance to irinotecan-based chemotherapy.

Paraneoplastic renal dysfunction in fly cancer models driven by inflammatory activation of stem cells

Tumors can induce systemic disturbances in distant organs, leading to physiological changes that enhance host morbidity. In Drosophila cancer models, tumors have been known for decades to cause hypervolemic "bloating" of the abdominal cavity. Here we use allograft and transgenic tumors to show that hosts display fluid retention associated with autonomously defective secretory capacity of fly renal tubules, which function analogous to those of the human kidney. Excretion from these organs is blocked by abnormal cells that originate from inappropriate activation of normally quiescent renal stem cells (RSCs). Blockage is initiated by IL-6-like oncokines that perturb renal water-transporting cells and trigger a damage response in RSCs that proceeds pathologically. Thus, a chronic inflammatory state produced by the tumor causes paraneoplastic fluid dysregulation by altering cellular homeostasis of host renal units.

Neuroendocrine Differentiation in Prostate Cancer Requires ASCL1.

Most patients with prostate adenocarcinoma develop resistance to therapies targeting the androgen receptor (AR). Consequently, a portion of these patients develop AR-independent neuroendocrine prostate cancer (NEPC), a rapidly progressing cancer with limited therapies and poor survival outcomes. Current research to understand the progression to NEPC suggests a model of lineage plasticity whereby AR-dependent luminal-like tumors progress towards an AR-independent NEPC state. Genetic analysis of human NEPC identified frequent loss of RB1 and TP53, and the loss of both genes in experimental models mediates the transition to a neuroendocrine lineage. Transcriptomics studies have shown that lineage transcription factors ASCL1 and NEUROD1 are present in NEPC. In this study, we modeled the progression of prostate adenocarcinoma to NEPC by establishing prostate organoids and subsequently generating subcutaneous allograft tumors from genetically-engineered mouse models harboring Cre-induced loss of Rb1 and Trp53 with Myc overexpression (RPM). These tumors were heterogeneous and displayed adenocarcinoma, squamous, and neuroendocrine features. ASCL1 and NEUROD1 were expressed within neuroendocrine-defined regions, with ASCL1 being predominant. Genetic loss of Ascl1 in this model did not decrease tumor incidence, growth, or metastasis; however, there was a notable decrease in neuroendocrine identity and an increase in basal-like identity. This study provides an in vivo model to study progression to NEPC and establishes the requirement for ASCL1 in driving neuroendocrine differentiation in prostate cancer.

Anti-angiogenic activity of a novel angiostatin-like plasminogen fragment produced by a bacterial metalloproteinase

Tumor growth depends on angiogenesis, a process by which new blood vessel are formed from pre-existing normal blood vessels. Proteolytic fragments of plasminogen, containing varying numbers of plasminogen kringle domains, collectively known as angiostatin, are a naturally occurring inhibitor of angiogenesis and inhibit tumor growth. We have developed an “affinity-capture reactor” that enables a single-step method for the production/purification of an angiostatin-like plasminogen fragment from human plasma using an immobilized bacterial metalloproteinase. The resulting fragment, named BL-angiostatin, contains one or two glycosyl chains and the N-terminal PAN module, which are not present in canonical angiostatins tested for cancer treatment. BL-angiostatin inhibited angiogenesis in vitro at 20 nM and the growth of both allograft and human xenograft tumors as well as lung metastasis of primary tumors mice at 0.3–10 mg kg−1. Derivatives of BL angiostatin lacking the PAN module or the terminal sialic acids in the glycosyl chains showed reduced anti-angiogenic activity in vivo, suggesting a role for these functions in activity, possibly via conferring a pharmacokinetic advantage to BL angiostatin compared to recombinant angiostatin lacking both features. These results highlight the potential of BL-angiostatin for therapeutic applications.

Obesity Induces Temporally Regulated Alterations in the Extracellular Matrix That Drive Breast Tumor Invasion and Metastasis.

Obesity is associated with increased incidence and metastasis of triple-negative breast cancer, an aggressive breast cancer subtype. The extracellular matrix (ECM) is a major component of the tumor microenvironment that drives metastasis. To characterize the temporal effects of age and high-fat diet (HFD)-driven weight gain on the ECM, we injected allograft tumor cells at 4-week intervals into mammary fat pads of mice fed a control or HFD, assessing tumor growth and metastasis and evaluating the ECM composition of the mammary fat pads, lungs, and livers. Tumor growth was increased in obese mice after 12 weeks on HFD. Liver metastasis increased in obese mice only at 4 weeks, and elevated body weight correlated with increased metastasis to the lungs but not the liver. Whole decellularized ECM coupled with proteomics indicated that early stages of obesity were sufficient to induce changes in the ECM composition. Obesity led to an increased abundance of the proinvasive ECM proteins collagen IV and collagen VI in the mammary glands and enhanced the invasive capacity of cancer cells. Cells of stromal vascular fraction and adipose stem and progenitor cells were primarily responsible for secreting collagen IV and collagen VI, not adipocytes. Longer exposure to HFD increased the invasive potential of ECM isolated from the lungs and liver, with significant changes in ECM composition found in the liver with short-term HFD exposure. Together, these data suggest that changes in the breast, lungs, and liver ECM underlie some of the effects of obesity on triple-negative breast cancer incidence and metastasis. Significance: Organ-specific extracellular matrix changes in the primary tumor and metastatic microenvironment are mechanisms by which obesity contributes to breast cancer progression.

Abstract PO-019: Ubiquitin E3 ligase OSTM1 regulates the cAMP/PKA/CREB pathway and suppresses B-cell malignancies

Abstract B-cell malignancies (BCM) often arise from genomic alterations introduced during different stages of B-cell development. Here, we performed a whole genome targeting CRISPR/Cas9 screen in the immortalized yet non-transformed IL-3-dependent Ba/F3 murine pro-B cell line to identify genes whose loss confers IL-3 independence, reasoning that these genes may function as tumor suppressor genes (TSGs) in BCM. One of the candidate genes is osteoporosis-associated transmembrane protein 1 (OSTM1), an E3 ubiquitin ligase whose loss-of-function causes autosomal recessive osteoporosis. Informatics analysis of patient data showed that OSTM1 is frequently deleted across BCM, which co-occurs with the deletion of a well-characterized TSG Cdkn2a. Genetic silencing of OSTM1 in Ba/F3 cells conferred IL-3-independent survival and proliferation, as well as splenomegaly, lymphadenopathy, and abnormal peripheral blood cell count, indicative of transformation in vivo. Using mass spectrometry screens, we identified phosphodiesterase 3B (PDE3B) as an interacting partner of OSTM1. OSTM1 ubiquitylates PDE3B and promotes its proteasomal degradation. The OSTM1-PDE3B interaction and PDE3B degradation are negatively regulated by OSTM1 N-glycosylation. As PDE3B catalyzes the conversion of cAMP to AMP, it negatively regulates the cAMP-dependent PKA/CREB/CREBBP tumor suppressive pathway. Indeed, overexpression of PDE3B accelerated cell proliferation while PDE3B silencing in OSTM1 KO cells reversed the transformation phenotype in cell lines and in mouse allograft tumors. Importantly, a strong correlation between OSTM1 disruption, increased PDE3B stability, and decreased PKA/CREB/CEBBP signaling was observed in cell lines and in BCM patient datasets. Finally, CD19-Cre mediated B-cell specific ablation of OSTM1 cooperated with CDKN2A deletion to drive spontaneous immature B-cell lymphomagenesis in mice. Collectively, these results indicate that OSTM1 is a novel TSG which targets PDE3B for proteasomal degradation. Loss of OSTM1 enhances PDE3B stability and abrogates the tumor-suppressive role of cAMP/CREB/CREBBP pathway which promotes BCM. Citation Format: Muhammad Usama Tariq, Julia Shen, Jaeyong Jung, Namratha Sheshadri, Junrong Yan, Rongrong Li, Kevin Lu, Tong Liu, Hong Li, Yue Lynn Wang, Ping Xie, Wei-Xing Zong. Ubiquitin E3 ligase OSTM1 regulates the cAMP/PKA/CREB pathway and suppresses B-cell malignancies [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-019.

Abstract PO-035: Genome-wide CRISPR knockout screen identifies sialylation as a tumor suppressive mechanism in B-cell malignancies

Abstract Ba/F3 is a murine pro-B cell line that is dependent on interleukin-3 (IL-3) for its survival and proliferation. It has been widely used for identifying oncogenes and mechanisms of drug-resistance. Using this cell line, we conducted a genome-wide CRISPR knockout screen to identify genes whose loss can confer IL-3 independence, hence may be implied as tumor suppressor genes (TSGs). We identified several genes that map to human chromosome 6q, a frequent deletion hotspot in B-cell malignancies, that may act as TSGs. One of these putative novel TSGs is Slc35a1, which encodes the membrane solute carrier that facilitates the transport of nucleotide sugar (CMP-sialic acid) into the Golgi apparatus for subsequent sialylation. Interestingly, in addition to Slc35a1, pathway analysis of hits from the CRISPR screen identified several genes in the sialic acid metabolism pathway, Nans, Cmas, and St3gal5. We confirmed that their silencing led to decreased protein sialyation and could confer IL-3-independent cell survival and growth in Ba/F3 cells. We continued to characterize Slc35a1 since its homozygous and heterozygous deletions are seen in 7-40% of different types of B-cell malignancies, including approximately 40% of patients with Diffuse Large B-cell Lymphoma. Slc35a1 deletion in murine and human cell-lines conferred cytokine-independent growth. Tail-vein injection of Slc35a1-silenced Ba/F3 cells resulted in allograft tumor growth presenting with splenomegaly and lymphadenopathy. Using Maackia amurensis lectin II (MAL II) to pull down sialylated proteins for mass spectrometry, we identified proteins that were differentially sialylated in Slc35a1 wild-type and knockout cells, among which are the α and β subunits of the integrin receptor, lymphocyte functional antigen 1 (LFA-1). As integrin desialylation can promote its constitutive activation, we are currently studying the impact of LFA-1 sialylation on B-cell function and transformation. We have also developed mouse models with B-cell specific ablation of Nans and Slc35a1 to further evaluate the role of sialylation on B-cell development and tumorigenesis in vivo. Overall, our study demonstrates that sialylation may function as a tumor suppressive mechanism and serve as a therapeutic target in B-cell cancers. Citation Format: Namratha Sheshadri, Rongrong Li, Muhammad Usama Tariq, Jianliang Shen, Jaeyong Jung, Junrong Yan, Kevin Lu, Zhiyuan Shen, Ping Xie, Wei-Xing Zong. Genome-wide CRISPR knockout screen identifies sialylation as a tumor suppressive mechanism in B-cell malignancies [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-035.

Novel engineered regulatory macrophages combined with ICIs for post-transplant recurrence after liver transplantation for HCC.

e14582 Background: Liver transplantation is the most effective treatment for early-stage hepatocellular carcinoma (HCC), but post-transplant recurrence limits the efficacy of surgery and affects the patient prognosis. Most of the current attempts to apply immune checkpoint inhibitors (ICIs) to posttransplant recurrence have resulted in either acute allograft rejection or tumor progression, which may be related to the disruption of the immune balance that maintains immune tolerance and enhances immune activation. Methods: Therefore, we constructed an engineered regulatory macrophages (Mregs) with anti-tumor capacity in the transplant setting using reprogrammed liposomes to precisely regulate the immune microenvironment of post-transplant recurrence, in order to create suitable conditions for the safe use of ICIs. Results: Experimentally, Mregs were shown to prolonged allograft survival without promoting tumor development. Through fusing with the Mregs membrane, reprogrammed liposomes insert an anti-CD47 lipid shell onto the surface, enhancing phagocytosis against tumor. The drug-loaded inner core entered into the cytoplasm to reprogram Mregs. Tomivosertib downregulated the expression of PD-L1 and attenuated the immunosuppressive effect. Pexidartinib inhibited the polarization of the M2 pathway and upregulated the expression of CD80, CD86, and MHC II, enhancing the ability of antigen presentation. Combination with anti-PD-L1 antibodies can enhance anti-tumor effect without accelerating allograft rejection. Both in vivo and in vitro results demonstrate that engineered Mregs provide conditions for the safe use of anti-PD-L1 antibodies in the context of transplantation. Conclusions: Engineered Mregs combined with ICIs enhances immunotherapeutic efficacy and provides a viable therapeutic approach for posttransplant recurrence after liver transplantation for HCC.

Isoalantolactone exerts anti-melanoma effects via inhibiting PI3K/AKT/mTOR and STAT3 signaling in cell and mouse models.

Although the anti-cancer activity of isoalantolactone (IATL) has been extensively studied, the anti-melanoma effects of IATL are still unknown. Here, we have investigated the anti-melanoma effects and mechanism of action of IATL. MTT and crystal violet staining assays were performed to detect the inhibitory effect of IATL on melanoma cell viability. Apoptosis and cell cycle arrest induced by IATL were examined using flow cytometry. The molecular mechanism of IATL was explored by Western blotting, confocal microscope analysis, molecular docking, and cellular thermal shift assay (CETSA). A B16F10 allograft mouse model was constructed to determine the anti-melanoma effects of IATL in vivo. The results showed that IATL exerted anti-melanoma effects in vitro and in vivo. IATL induced cytoprotective autophagy in melanoma cells by inhibiting the PI3K/AKT/mTOR signaling. Moreover, IATL inhibited STAT3 activation both in melanoma cells and allograft tumors not only by binding to the SH2 domain of STAT3 but also by suppressing the activity of its upstream kinase Src. These findings demonstrate that IATL exerts anti-melanoma effects via inhibiting the STAT3 and PI3K/AKT/mTOR signaling pathways, and provides a pharmacological basis for developing IATL as a novel phytotherapeutic agent for treating melanoma clinically.

Abstract 3969: Alzheimer's disease-associated amyloid precursor protein improves anti-tumor immunity by inhibiting ceramide-mediated lethal mitophagy and restoring mitochondrial fumarate metabolism in aging T-cells

Abstract The association between Alzheimer’s disease (AD) and lower cancer incidence has been observed in several epidemiological studies without much mechanistic insight. We aimed to understand this cancer resistance mechanism linked to AD and exploit it for broader cancer therapies. A hallmark of AD is the accumulation of amyloid precursor protein (APP) in neuronal mitochondria, inhibiting mitophagy, abrogating the clearance of dysfunctional mitochondria, and contributing to AD pathology. In addition to neurons, mitophagy plays an important role in T-cells, where excessive ceramide-dependent mitophagy in aging T-cells inhibits their energy metabolism and limits their anti-tumor functions. Thus, attenuation of ceramide-dependent mitophagy plays a favorable role in enhancing aging T-cell anti-tumor function. Given that APP is also processed in T-cells, we hypothesized that APP accumulates aberrantly in aging Alzheimer’s T-cells, inhibiting mitophagy and enhancing T-cell anti-tumor activity. Employing techniques of lipidomics, metabolomics, imaging, molecular biology, and genetic models, we show that AD T-cells from a mouse model and human patients are protected against aging-associated ceramide-dependent mitophagy, which enhanced their secretion of inflammatory anti-tumor cytokines. The excessive localization of APP to the T-cell mitochondria, where it inhibits the accumulation of ceramide synthase 6 (CerS6) and C14/C16-ceramides, attenuated the levels of mitophagy and protected the Alzheimer’s T-cells from metabolic defects observed in aging, namely protecting against the exhaustion of fumarate pools. Both in vitro and in vivo re-supplementation of fumarate to aging WT mouse T-cells effectively rescued their viability, cytokine production, and tumor killing capacity both in vitro and in adoptive T-cell transfers to mice with melanoma, functionally mimicking the AD phenotype. The fumarate supplementation also led to a decrease in ceramide-dependent mitophagy in the WT aging T-cells, indicating an interplay between ceramide-dependent mitophagy and fumarate metabolism which contributes to the protection of the anti-tumor function in aging AD T-cells. Moreover, we show that Alzheimer’s mice that express APP in the brain and T-cells, but not AD mice that express APP only in the brain, are protected against the growth of both carcinogen-induced and allografted tumors. Finally, transfer of mitochondria from AD T-cells to aging WT T-cells improved their metabolic fitness and anti-cancer functions. In summary, we show that AD-associated APP improves anti-tumor immunity of aging T-cells by inhibiting ceramide-mediated lethal mitophagy and restoring mitochondrial fumarate metabolism. These data provide a novel mechanism to explain reduced cancer incidences in AD patients. Citation Format: Mohamed Faisal Kassir, Han G. Lee, Natalia Oleinik, Wyatt Wofford, Chase Walton, F. Cansu Atilgan, Paramita Chakraborty, Kubra Calisir, Ozgur Sahin, Shikhar Mehrotra, Besim Ogretmen. Alzheimer's disease-associated amyloid precursor protein improves anti-tumor immunity by inhibiting ceramide-mediated lethal mitophagy and restoring mitochondrial fumarate metabolism in aging T-cells [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 3969.

Abstract 7300: Synergistic inhibition of prostate cancer growth by the combination of xanthohumol and ursolic acid

Abstract Within the United States, prostate cancer (PCa) is the most commonly diagnosed non-cutaneous cancer, and approximately 1 in 8 men will receive a diagnosis in their lifetime. As the second leading cause of cancer deaths in American men, about 1 in 41 will die because of prostate cancer. The World Health Organization has suggested that approximately one-third of all cancer deaths are preventable, and one of the suggested means of prevention is through the increased consumption of fruits and vegetables. Fruits and vegetables contain natural compounds known as phytochemicals which possess biological activity including some anti-cancer effects. By adapting the ATP-bioluminescence assay for cell viability, our laboratory screened a natural compound library to find combinations of these compounds that may synergistically inhibit PCa cell viability. One of the combinations to emerge from this screening process was xanthohumol (XAN) and ursolic acid (UA). We examined the ability of the combination to inhibit tumor growth in a mouse HMVP2 PCa allograft tumor model. Mice were fed diets supplemented with either XAN (0.2%) or UA (0.2%) alone or the combination. Mice on the combination diet showed a significant inhibition of HMVP2 PCa tumor growth compared to mice on the single-agent diets. In addition, in vitro studies were performed to support the allograft tumor study and to further examine the mechanism of action of the combination. A cell survival assay utilizing six different PCa cell lines indicated that the combination of XAN and UA synergistically inhibited the survival of all cell lines as calculated by the Bliss index. Mechanistic studies performed in PCa cells indicate that the combination of XAN and UA inhibits oncogenic signaling better than either agent alone. Additionally, unbiased metabolomics revealed that the tumors from mice fed the combination diet had significantly less S-adenosyl-L-methionine compared to all other groups. Overall, 13 metabolic pathways were significantly different in the tumors from mice fed the combination diet compared to all other groups. The current data suggest that using a combination of XAN and UA may be a valuable chemopreventive strategy for PCa in humans. Funding: This work was supported by NIH Grant CA228404. Citation Format: Rachel Clark, Achinto Saha, Chelsea A. Friedman, Ruggiero Gorgoglione, Stefano Tiziani, John DiGiovanni. Synergistic inhibition of prostate cancer growth by the combination of xanthohumol and ursolic acid [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 7300.

Combined positron emission tomography‐guided modified black phosphorus nanosheet‐based photothermal therapy and anti programmed cell death protein ligand 1 therapy

AbstractBackgroundPatients with cold tumors gain limited benefits from immune checkpoint blockade (ICB) therapy owing to low programmed cell death protein ligand 1 (PD‐L1) expression and minimal immune cell infiltration. Mild photothermal therapy (PTT) using black phosphorus nanosheets (BPNSs) is a promising approach to enhance the efficacy of ICB therapy. However, to ensure that BPNS‐based PTT‐enhanced ICB therapy is clinically adaptable, a noninvasive, bedside‐accessible imaging tool capable of monitoring the status of PD‐L1 is imperative. We demonstrated that positron emission tomography (PET) using [64Cu]HKP2202 precisely delineated PD‐L1 expression in tumors receiving PTT.MethodsBPNSs were modified with polyethylene glycol to prepare BPNS@PEG, which were then characterized. MC38 cells and tumor allografts were treated with BPNS@PEG followed by 808 nm near‐infrared light exposure. PET using [64Cu]HKP2202 was performed to monitor PD‐L1 expression in vivo. We also evaluated whether the efficacy of ICB therapy improved after delivering BPNS@PEG‐based PTT.ResultsBPNS@PEG had a well‐defined lamellar structure with clear edges and an average size of 150 nm. PET and Western blotting assays indicated that PD‐L1 expression was upregulated after BPNS@PEG and NIR‐light treatment. Notably, the antitumor effect of anti PD‐L1 therapy was enhanced in mice treated with BPNS@PEG‐based PTT.ConclusionsBPNS@PEG had the capacity to convert cold tumors into hot tumors to facilitate the efficacy of ICB therapy. Importantly, the complementary diagnostic PET radiotracer targeting PD‐L1 allowed real‐time monitoring of PD‐L1 expression in the tumor microenvironment to guide ICB administration, holding great potential to achieve efficient and precise tumor immunotherapy.

CD5 blockade, a novel immune checkpoint inhibitor, enhances T cell anti-tumour immunity and delays tumour growth in mice harbouring poorly immunogenic 4T1 breast tumour homografts.

CD5 is a member of the scavenger receptor cysteine-rich superfamily that is expressed on T cells and a subset of B cells (B1a) cell and can regulate the T cell receptor signaling pathway. Blocking CD5 function may have therapeutic potential in treatment of cancer by enhancing cytotoxic T lymphocyte recognition and ablation of tumour cells. The effect of administering an anti-CD5 antibody to block or reduce CD5 function as an immune checkpoint blockade to enhance T cell anti-tumour activation and function in vivo has not been explored. Here we challenged mice with poorly immunogenic 4T1 breast tumour cells and tested whether treatment with anti-CD5 monoclonal antibodies (MAb) in vivo could enhance non-malignant T cell anti-tumour immunity and reduce tumour growth. Treatment with anti-CD5 MAb resulted in an increased fraction of CD8+ T cells compared to CD4+ T cell in draining lymph nodes and the tumour microenvironment. In addition, it increased activation and effector function of T cells isolated from spleens, draining lymph nodes, and 4T1 tumours. Furthermore, tumour growth was delayed in mice treated with anti-CD5 MAb. These data suggest that use of anti-CD5 MAb as an immune checkpoint blockade can both enhance activation of T cells in response to poorly immunogenic antigens and reduce tumour growth in vivo. Exploration of anti-CD5 therapies in treatment of cancer, alone and in combination with other immune therapeutic drugs, is warranted.

HMGA1 drives chemoresistance in esophageal squamous cell carcinoma by suppressing ferroptosis

Chemotherapy is a primary treatment for esophageal squamous cell carcinoma (ESCC). Resistance to chemotherapeutic drugs is an important hurdle to effective treatment. Understanding the mechanisms underlying chemotherapy resistance in ESCC is an unmet medical need to improve the survival of ESCC. Herein, we demonstrate that ferroptosis triggered by inhibiting high mobility group AT-hook 1 (HMGA1) may provide a novel opportunity to gain an effective therapeutic strategy against chemoresistance in ESCC. HMGA1 is upregulated in ESCC and works as a key driver for cisplatin (DDP) resistance in ESCC by repressing ferroptosis. Inhibition of HMGA1 enhances the sensitivity of ESCC to ferroptosis. With a transcriptome analysis and following-up assays, we demonstrated that HMGA1 upregulates the expression of solute carrier family 7 member 11 (SLC7A11), a key transporter maintaining intracellular glutathione homeostasis and inhibiting the accumulation of malondialdehyde (MDA), thereby suppressing cell ferroptosis. HMGA1 acts as a chromatin remodeling factor promoting the binding of activating transcription factor 4 (ATF4) to the promoter of SLC7A11, and hence enhancing the transcription of SLC7A11 and maintaining the redox balance. We characterized that the enhanced chemosensitivity of ESCC is primarily attributed to the increased susceptibility of ferroptosis resulting from the depletion of HMGA1. Moreover, we utilized syngeneic allograft tumor models and genetically engineered mice of HMGA1 to induce ESCC and validated that depletion of HMGA1 promotes ferroptosis and restores the sensitivity of ESCC to DDP, and hence enhances the therapeutic efficacy. Our finding uncovers a critical role of HMGA1 in the repression of ferroptosis and thus in the establishment of DDP resistance in ESCC, highlighting HMGA1-based rewiring strategies as potential approaches to overcome ESCC chemotherapy resistance.Schematic depicting that HMGA1 maintains intracellular redox homeostasis against ferroptosis by assisting ATF4 to activate SLC7A11 transcription, resulting in ESCC resistance to chemotherapy.

The heterobivalent (SSTR2/albumin) radioligand [67Cu]Cu-NODAGA-cLAB4-TATE enables efficient somatostatin receptor radionuclide theranostics.

Somatostatin type 2 receptor (SSTR2) radionuclide therapy using β- particle-emitting radioligands has entered clinical practice for the treatment of neuroendocrine neoplasms (NENs). Despite the initial success of [177Lu]Lu‑DOTA-TATE, theranostic SSTR2 radioligands require improved pharmacokinetics and enhanced compatibility with alternative radionuclides. Consequently, this study evaluates the pharmacokinetic effects of the albumin-binding domain cLAB4 on theranostic performance of copper‑67-labeled NODAGA-TATE variants in an SSTR2-positive mouse pheochromocytoma (MPC) model. Methods: Binding, uptake, and release of radioligands as well as growth-inhibiting effects were characterized in cells grown as monolayers and spheroids. Tissue pharmacokinetics, absorbed tumor doses, and projected human organ doses were determined from quantitative SPECT imaging in a subcutaneous tumor allograft mouse model. Treatment effects on tumor growth, leukocyte numbers, and renal albumin excretion were assessed. Results: Both copper‑64- and copper‑67-labeled versions of NODAGA-TATE and NODAGA-cLAB4‑TATE showed similar SSTR2 binding affinity, but faster release from tumor cells compared to the clinical reference [177Lu]Lu‑DOTA-TATE. The bifunctional SSTR2/albumin-binding radioligand [67Cu]Cu‑NODAGA-cLAB4‑TATE showed both an improved uptake and prolonged residence time in tumors resulting in equivalent treatment efficacy to [177Lu]Lu‑DOTA-TATE. Absorbed doses were well tolerated in terms of leukocyte counts and kidney function. Conclusion: This preclinical study demonstrates therapeutic efficacy of [67Cu]Cu‑NODAGA-cLAB4‑TATE in SSTR2-positive tumors. As an intrinsic radionuclide theranostic agent, the radioligand provides stable radiocopper complexes and high sensitivity in SPECT imaging for prospective determination and monitoring of therapeutic doses in vivo. Beyond that, copper‑64- and copper‑61-labeled versions offer possibilities for pre- and post-therapeutic PET. Therefore, NODAGA-cLAB4-TATE has the potential to advance clinical use of radiocopper in SSTR2-targeted cancer theranostics.

Arctigenin inhibits the progression of colorectal cancer through epithelial-mesenchymal transition via PI3K/Akt/mTOR signaling pathway.

Colorectal cancer (CRC) is a significant disease worldwide, with high mortality rates. Conventional treatment methods often lead to metastasis and drug resistance, highlighting the need to explore new drugs and their potential molecular mechanisms. In this study, we investigated the effects of arctigenin on CRC cell proliferation, migration, invasion, apoptosis, and related protein expression, as well as its potential molecular mechanisms. The CCK-8 assay, transwell migration and invasion assays, flow cytometry, immunoblotting and immunofluorescence staining, western blot and an allograft tumor transplantation model was used. Our study revealed that arctigenin effectively inhibited CRC cell proliferation, migration, and invasion in a dose-dependent manner, while also inducing apoptosis. At the molecular level, arctigenin significantly downregulated the expressions of PCNA, Bcl2, MMP-2, and MMP-9 and upregulated the expressions of Bax and cleaved caspase-3. Additionally, arctigenin demonstrated the ability to inhibit the epithelial-mesenchymal transition (EMT) process by upregulating E-cadherin and downregulating mesenchymal markers, such as N-cadherin, Vimentin, Snail, and Slug. Furthermore, arctigenin could inhibit the activation of the PI3K-AKT-mTOR signaling pathway, which has been implicated in cancer progression. In vivo experiments also showed that arctigenin significantly reduced tumor volume and size compared to the control group, with no significant adverse effects on the liver. This is the first study to elucidate the mechanism by which arctigenin inhibits colorectal cancer metastasis through the PI3K-AKT-mTOR signaling pathway by suppressing the EMT process at the molecular level.

Liver cancer stem cell dissemination and metastasis: uncovering the role of NRCAM in hepatocellular carcinoma

BackgroundLiver cancer stem cells (LCSCs) play an important role in hepatocellular carcinoma (HCC), but the mechanisms that link LCSCs to HCC metastasis remain largely unknown. This study aims to reveal the contributions of NRCAM to LCSC function and HCC metastasis, and further explore its mechanism in detail.Methods117 HCC and 29 non-HCC patients with focal liver lesions were collected and analyzed to assess the association between NRCAM and HCC metastasis. Single-cell RNA sequencing (scRNA-seq) was used to explore the biological characteristics of cells with high NRCAM expression in metastatic HCC. The role and mechanism of NRCAM in LCSC dissemination and metastasis was explored in vitro and in vivo using MYC-driven LCSC organoids from murine liver cells.ResultsSerum NRCAM is associated with HCC metastasis and poor prognosis. A scRNA-seq analysis identified that NRCAM was highly expressed in LCSCs with MYC activation in metastatic HCC. Moreover, NRCAM facilitated LCSC migration and invasion, which was confirmed in MYC-driven LCSC organoids. The in vivo tumor allografts demonstrated that NRCAM mediated intra-hepatic/lung HCC metastasis by enhancing the ability of LCSCs to escape from tumors into the bloodstream. Nrcam expression inhibition in LCSCs blocked HCC metastasis. Mechanistically, NRCAM activated epithelial-mesenchymal transition (EMT) and metastasis-related matrix metalloproteinases (MMPs) through the MACF1 mediated β-catenin signaling pathway in LCSCs.ConclusionsLCSCs typified by high NRCAM expression have a strong ability to invade and migrate, which is an important factor leading to HCC metastasis.

Effect of omega-3 fatty acid diet on prostate cancer progression and cholesterol efflux in tumor-associated macrophages-dependence on GPR120.

Preclinical and clinical translational research supports the role of an ω-3 fatty acid diet for prostate cancer prevention and treatment. The anti-prostate cancer effects of an ω-3 diet require a functional host g-protein coupled receptor 120 (GPR120) but the underlying effects on the tumor microenvironment and host immune system are yet to be elucidated. Friend leukemia virus B (FVB) mice received bone marrow from green fluorescent protein (GFP) labeled GPR120 wild-type (WT) or knockout (KO) mice followed by implanting Myc-driven mouse prostate cancer (MycCap) allografts and feeding an ω-3 or ω-6 diet. Tumor associated immune cells were characterized by flow cytometry, and CD206+ tumor infiltrating M2-like macrophages were isolated for gene expression studies. MycCap prostate cancer cell conditioned medium (CM) was used to stimulate murine macrophage cells (RAW264.7) and bone marrow-derived (BMD) macrophages to study the effects of docosahexanoic acid (DHA, fish-derived ω-3 fatty acid) on M2 macrophage function and cholesterol metabolism. The bone marrow transplantation study showed that an ω-3 as compared to an ω-6 diet inhibited MycCaP allograft tumor growth only in mice receiving GPR120 WT but not GPR120 KO bone marrow. In the ω-3 group, GPR120 WT BMD M2-like macrophages infiltrating the tumor were significantly reduced in number and gene expression of cholesterol transporters Abca1, Abca6, and Abcg1. RAW264.7 murine macrophages and BMDMs exposed to MycCaP cell CM had increased gene expression of cholesterol transporters, depleted cholesterol levels, and were converted to the M2 phenotype. These effects were inhibited by DHA through the GPR120 receptor. Host bone marrow cells with functional GPR120 are essential for the anticancer effects of dietary ω-3 fatty acids, and a key target of the ω-3 diet are the M2-like CD206+ macrophages. Our preclinical findings provide rationale for clinical trials evaluating ω-3 fatty acids as a potential therapy for prostate cancer through inhibition of GPR120 functional M2-like macrophages.

Design, synthesis and biological evaluation of novel dihydroquinolin-4(1H)-one derivatives as novel tubulin polymerization inhibitors

A series of novel dihydroquinolin-4(1H)-one derivatives targeting colchicine binding site on tubulin were designed, synthesized and evaluated as anticancer agents. The most potent compound 6t showed remarkable antiproliferative activities against four cancer cell lines with IC50 values among 0.003–0.024 μM and tubulin polymerization inhibitory activity (IC50 = 3.06 μM). Further mechanism studies revealed that compound 6t could induce K562 cells apoptosis and arrest at the G2/M phase. Meanwhile, 6t significantly inhibited migration and invasion of MDA-MB-231 cells, and disrupted the angiogenesis in human umbilical vein endothelial cells (HUVECs) in vitro. In addition, compound 6t inhibited tumor growth in H22 allograft tumor model with a tumor growth inhibition (TGI) rate of 63.3 % (i.v., 20 mg/kg per day) without obvious toxicity. Collectively, these results indicated that compound 6t was a novel tubulin polymerization inhibitor with potent anticancer properties in vitro and in vivo.

TAK1 is an essential kinase for STING trafficking

The translocation of stimulator of interferon genes (STING) from the endoplasmic reticulum (ER) to the ER-Golgi intermediate compartment (ERGIC) enables its activation. However, the mechanism underlying the regulation of STING exit from the ER remains elusive. Here, we found that STING induces the activation of transforming growth factor beta-activated kinase 1 (TAK1) prior to STING trafficking in a TAK1 binding protein 1 (TAB1)-dependent manner. Intriguingly, activated TAK1 directly mediates STING phosphorylation on serine 355, which facilitates its interaction with STING ER exit protein (STEEP) and thereby promotes its oligomerization and translocation to the ERGIC for subsequent activation. Importantly, activation of TAK1 by monophosphoryl lipid A, a TLR4 agonist, boosts cGAMP-induced antitumor immunity dependent on STING phosphorylation in a mouse allograft tumor model. Taken together, TAK1 was identified as a checkpoint for STING activation by promoting its trafficking, providing a basis for combinatory tumor immunotherapy and intervention in STING-related diseases.