Squamous Cell Carcinoma Xenografts Research Articles

SMG-1 serves as a prognostic indicator for the radiotherapy response in head and neck squamous cell carcinoma xenografts and patients.

SMG-1 serves as a prognostic indicator for the radiotherapy response in head and neck squamous cell carcinoma xenografts and patients.

Loss of CELF2 promotes skin tumorigenesis and increases drug resistance.

CELF2 belongs to the CELF RNA-binding protein family and exhibits antitumor activity in various tumor models. Analysis of the pan-cancer TCGA database reveals that CELF2 expression strongly correlates with favorable prognosis among cancer patients. The function of CELF2 in nonmelanoma skin cancer has not been studied. We used shRNA-mediated knockdown (KD) of CELF2 expression in human squamous cell carcinoma (SCC) cells to investigate how CELF2 impacted SCC cell proliferation, survival, and xenograft tumor growth. We determined CELF2 expression in human SCC tissues and adjacent normal skin using immunofluorescence staining. Additionally, we investigated the changes in CELF2 and its target gene expression during UV-induced and chemical-induced skin tumorigenesis by western blotting. CELF2 KD significantly increased SCC cell proliferation, colony growth, and SCC xenograft tumor growth in immunodeficient mice. CELF2 KD in SCC cells led to activation of KRT80 and GDF15, which can potentially promote cell proliferation and tumor growth. While control SCC cells were sensitive to anticancer drugs such as doxorubicin, SCC cells with CELF2 KD became resistant to drug-induced tumor growth retardation. Finally, we found CELF2 expression diminished during both UV- and chemical-induced skin tumorigenesis in mice, consistent with reduced CELF2 expression in human SCC tumors compared to adjacent normal skin. This study shows for the first time that CELF2 loss occurs during skin tumorigenesis and increases drug resistance in SCC cells, highlighting the possibility of targeting CELF2-regulated pathways in skin cancer prevention and therapies.

Combination of vitamin D and photodynamic therapy enhances immune responses in murine models of squamous cell skin cancer

Improved treatment outcomes for non-melanoma skin cancers can be achieved if Vitamin D (Vit D) is used as a neoadjuvant prior to photodynamic therapy (PDT). However, the mechanisms for this effect are unclear. Vit D elevates protoporphyrin (PpIX) levels within tumor cells, but also exerts immune-modulatory effects. Here, two murine models, UVB-induced actinic keratoses (AK) and human squamous cell carcinoma (A431) xenografts, were used to analyze the time course of local and systemic immune responses after PDT ± Vit D. Fluorescence immunohistochemistry of tissues and flow analysis (FACS) of blood were employed. In tissue, damage-associated molecular patterns (DAMPs) were increased, and infiltration of neutrophils (Ly6G+), macrophages (F4/80+), and dendritic cells (CD11c+) were observed. In most cases, Vit D alone or PDT alone increased cell recruitment, but Vit D + PDT showed even greater recruitment effects. Similarly for T cells, increased infiltration of total (CD3+), cytotoxic (CD8+) and regulatory (FoxP3+) T-cells was observed after Vit D or PDT, but the increase was even greater with the combination. FACS analysis revealed a variety of interesting changes in circulating immune cell levels. In particular, neutrophils decreased in the blood after Vit D, consistent with migration of neutrophils into AK lesions. Levels of cells expressing the PD-1+ checkpoint receptor were reduced in AKs following Vit D, potentially counteracting PD-1+ elevations seen after PDT alone. In summary, Vit D and ALA-PDT, two treatments with individual immunogenic effects, may be advantageous in combination to improve treatment efficacy and management of AK in the dermatology clinic.

STAT3-EphA7 axis contributes to the progression of esophageal squamous cell carcinoma

Background Our previous study has revealed that EphA7 was upregulated in patient-derived esophageal squamous cell carcinoma (ESCC) xenografts with hyper-activated STAT3, but its mechanism was still unclear. Materials and methods To assess the association between EphA7 and STAT3, western blotting, immunofluorescence, ChIP assay, and qRT-PCR were conducted. Truncated mutation and luciferase assay were performed to examine the promoter activity of EphA7. CCK-8 assay and colony formation were performed to assess the proliferation of ESCC. Cell-derived xenograft models were established to evaluate the effects of EphA7 on ESCC tumor growth. RNA-seq analyses were used to assess the effects of EphA7 on related signals. Results In this study, EphA7 was found upregulated in ESCC cell lines with high STAT3 activation, and immunofluorescence also showed that EphA7 was co-localized with phospho-STAT3 in ESCC cells. Interestingly, suppressing STAT3 activation by the STAT3 inhibitor Stattic markedly inhibited the protein expression of EphA7 in ESCC cells, in contrast, activation of STAT3 by IL-6 obviously upregulated the protein expression of EphA7. Moreover, the transcription of EphA7 was also mediated by the activation of STAT3 in ESCC cells, and the −2000∼−1500 region was identified as the key promoter of EphA7. Our results also indicated that EphA7 enhanced the cell proliferation of ESCC, and silence of EphA7 significantly suppressed ESCC tumor growth. Moreover, EphA7 silence markedly abolished STAT3 activation-derived cell proliferation of ESCC. Additionally, RNA-seq analyses indicated that several tumor-related signaling pathways were significantly changed after EphA7 downregulation in ESCC cells. Conclusion Our results showed that the transcriptional expression of EphA7 was increased by activated STAT3, and the STAT3 signaling may act through EphA7 to promote the development of ESCC.

A pH-Activatable Nanoprobe Labels Diverse Histologic Subtypes of Human Lung Cancer During Resection.

Intraoperative molecular imaging (IMI) uses tumor-targeted optical contrast agents to improve identification and clearance of cancer during surgery. Recently, pH-activatable contrast agents have been developed but none has been tested in lung cancer. Here, we report the successful clinical translation of pegsitacianine (ONM-100), a pH-activatable nanoprobe, for fluorescence-guided lung cancer resection. We first characterized the pH setpoint for pegsitacianine fluorescence activation in vitro. We then optimized the specificity, dosing, and timing of pegsitacianine in murine flank xenograft models of lung adenocarcinoma and squamous cell carcinoma. Finally, we tested pegsitacianine in humans undergoing lung cancer surgery as part of an ongoing phase 2 trial. We found that the fluorescence activation of pegsitacianine occurred below physiologic pH in vitro. Using preclinical models of lung cancer, we found that the probe selectively labeled both adenocarcinoma and squamous cell carcinoma xenografts (mean tumor-to-background ratio [TBR] > 2.0 for all cell lines). In the human pilot study, we report cases in which pegsitacianine localized pulmonary adenocarcinoma and pulmonary squamous cell carcinoma (TBRs= 2.7 and 2.4) in real time to illustrate its successful clinical translation and potential to improve surgical management. This translational study demonstrates the feasibility of pegsitacianine as an IMI probe to label the two most common histologic subtypes of human lung cancer.

Transcutaneous carbon dioxide application suppresses the expression of cancer-associated fibroblasts markers in oral squamous cell carcinoma xenograft mouse model.

Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer. Cancer-associated fibroblasts (CAFs) are the main stromal cells in the tumor microenvironment (TME). As CAFs promote tumor progression and hypoxia in the TME, regulating the conversion of normal fibroblasts (NFs) into CAFs is essential for improving the prognosis of patients with OSCC. We have previously reported the antitumor effects of transcutaneous carbon dioxide (CO2) application in OSCC. However, the effects of reducing hypoxia in the TME remain unclear. In this study, we investigated whether CO2 administration improves the TME by evaluating CAFs marker expression. Human OSCC cells (HSC-3) and normal human dermal fibroblasts (NHDF) were coinjected subcutaneously into the dorsal region of mice. CO2 gas was applied twice a week for 3 weeks. The tumors were harvested six times after transcutaneous CO2 application. The expression of CAFs markers (α-SMA, FAP, PDPN, and TGF-β) were evaluated by using real-time polymerase chain reaction and immunohistochemical staining. The expression of α-SMA, FAP, PDPN, and TGF-β was significantly increased over time after co-injection. In the CO2-treated group, tumor growth was significantly suppressed after treatment initiation. In addition, the mRNA expression of these markers was significantly inhibited. Furthermore, immunohistochemical staining revealed a significant decrease in the protein expression of all CAFs markers in the CO2-treated group. We confirmed that transcutaneous CO2 application suppressed CAFs marker expression and tumor growth in OSCC xenograft mouse model.

Intravenous Delivery of miR-21 Inhibitor in Mice With Head and Neck Squamous Cell Carcinoma.

Synthetic miRNA inhibitors have recently attracted considerable interest as potential therapeutic agents for head and neck squamous cell carcinoma. However, due to the lack of evidence, no attempts have been made to deliver these inhibitors intravenously for squamous cell carcinoma. This study investigated whether intravenous administration of a miR-21 inhibitor with lipid nanoparticles could suppress HNSCC in xenograft mice. Head and neck squamous cell carcinoma xenograft mice were intravenously injected with Invivofectamine 3.0® containing either a miR-21 inhibitor or a control inhibitor, using a modified protocol for nucleic acid encapsulation. Quantitative PCR was used to measure the expression level of intratumoral miR-21. And TdT-mediated dUTP nick-end labeling (TUNEL) immunohistochemistry was used to assess cell death. Intravenous injection of miR-21 inhibitor significantly inhibited head and neck squamous cell carcinoma growth and miR-21 expression in tumor tissue compared to the control inhibitor. TUNEL assay showed significant apoptosis of tumor cells after intravenous administration of miR-21 inhibitor. Intravenous delivery of a miR-21 inhibitor with lipid nanoparticles is a promising approach for miRNA-targeted therapy of head and neck squamous cell carcinoma.

Abstract LB111: Epigenetic mechanism underlying pathogenesis of skin photo-damage and actinic keratoses

Abstract Chronic skin exposure to solar ultraviolet B (UVB) radiation causes cutaneous inflammation, sun-burns and pre-malignant lesions known as Actinic Keratosis (AK). About 20% of AKs may progress to highly invasive malignant lesions known as Squamous Cell Carcinoma (SCC). These lesions carry multiple mutations in oncogenes and/or tumor suppressor genes. However, the molecular mechanism underlying AK is not incompletely defined. Here, we demonstrate that cutaneous UVB exposure leads to activation of bromodomain-4 (BRD4), an epigenetic modifier. We observed enhanced expression of BRD4 and its hyper-acetylation marks, H3K18ac and H3K27ac in AK, accompanied by elevated transcription of BRD4-target genes: MMP13, CXCL9, IL33, Apol19b, IL19 and Oasl2. Using confocal microscopy, we observed enhanced nuclear accumulation of BRD4 and H3K27ac in the sun-exposed lesional skin, AK and in SCC tissues from patients. Ptch+/−/SKH1 mice irradiated with to chronic UVB manifest similar molecular alterations in highly photo-damaged skin. By manipulating BRD4 expression in human skin SCC A431 cells, we identified two important BRD4-regulated signaling pathways, which may be involved in the molecular pathogenesis of AKs and SCCs. BRD4-mediated inflammatory cytokine signaling is regulated by EZH2-SOCS3-NFkB/STAT3 axis while Myc-Survivin-CDK4-E2F1 axis drives tumor cell hyper-proliferation. Co-localization of BRD4/H3K27ac and Survivin-E2F1 was contiguous from sun-burn skin to AK and SCC. We observed enhanced copy number of Myc in human AK lesional skin and associated SCCs. We also show that Myc-Survivin-CDK4-E2F1 axis is critical in driving the progression of tumor keratinocytes to highly invasive and fast growing SCCs. This signaling acts by enhancing the expression epithelial-mesenchymal transition transcription factors and other proteins involved in UVB-induced SCC development. 9cUAB30, a RXR agonist developed by us blocked BRD4 activation and its downstream signaling in A431 and SCC13 cells. Oral administration of 9cUAB30 abrogates UVB-induced inflammation and keratinocyte hyper-proliferation in murine skin in BRD4 dependent manner. 9cUAB30 treatment attenuates growth of human SCC xenograft tumors in highly immune-suppressed mice, a response which was significantly more profound when the drug was administered with a survivin inhibitor, LQZ-7I. These translational studies demonstrate that UVB-induced cutaneous BRD4 drives AK pathogenesis and their progression to invasive SCCs. Citation Format: Mahendra P. Kashyap, Rajesh Sinha, Rubina Shafi, Venkatram R. Atigadda, Craig A. Elmets, Mohammad Athar. Epigenetic mechanism underlying pathogenesis of skin photo-damage and actinic keratoses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB111.

Abstract 363: Modelling cancer cachexia using a comprehensive panel of heterogeneous esophageal squamous cell carcinoma xenografts for therapeutic application

Abstract Background: Esophageal squamous cell carcinoma (ESCC) is one of deadliest esophageal malignancies in Asia. Cancer cachexia is a multifactorial body wasting syndrome and is one of the major causes of death in late-stage patients (1). It is characterized by skeletal muscle loss and atrophy, adipose and body wasting, and metabolic dysregulations and stress (1). ESCC is one of the cancer types with the highest morbidity and mortality rates due to cancer cachexia (2). However, there are few effective and approved treatments targeting cancer cachexia. Currently, cancer cachexia animal models are highly limited with few cancer types available as a pre-clinical drug testing platform, with none related to esophageal carcinoma (3). Results: Here we have developed a panel of ESCC patient-derived organoid xenograft (PDOX) mouse model based on our latest patient-derived organoid cultures originating from Hong Kong patients. Our immunodeficient mouse-based PDOX model resembles different cachexic phenotypes that cell line-derived xenograft lacks, including body (skeletal muscle and adipose tissue) wasting and functional signs (e.g. lack of body movement, reduced muscle strength, and loss of appetite). The slow-proliferating characteristic of the ESCC PDOX allows development of a cachexic condition before humane endpoint. Furthermore, our full panel of PDOX lines allowed us to identify the full spectrum, differential cachexia severity across mice bearing different PDOXs, indicating a heterogenous nature. We have comprehensively characterized the cachexia phenotypes and performed proof-of-concept preclinical drug tests using established anti-cachexic drugs; treated PDOX-bearing mice have improved body condition and function compared to the control, which were further verified by histological examinations of tissue samples. Molecular analysis showed that several classic cachexia related gene expressions were down-regulated in isolated liver (Il1b) and inguinal adipose tissues (adipose triglyceride lipase). Conclusions: Our ESCC PDOX model potentially serves as a great pre-clinical cachexia-targeted drug testing platform, providing more treatment options for ESCC patients for better disease management. Acknowledgement: We acknowledge the Research Grants Council (TRS T12-701/17-R to MLL) and the Food and Health Bureau (HMRF 06171566 to VZY) of Hong Kong Special Administrative Region for funding supports. We acknowledge DSMZ for the KYSE cell lines. We acknowledge the HKUMed-CPOS for providing imaging facilities.

Efficacy of PP121 in primary and metastatic non‑small cell lung cancers.

Tyrosine kinase inhibitors are a clinically standard treatment option for non-small cell lung cancers (NSCLCs), the leading cause of cancer-related deaths in the US. These targeted agents include first, second and third generation tyrosine kinase inhibitors; however, these lack clinical efficacy in the treatment of NSCLC due to intrinsic and acquired resistance. This resistance may be a result of genetic aberrations in oncogenic signaling mediators of divergent pathways. The present study aimed to investigate a novel dual tyrosine kinase and PI3K inhibitor, PP121, as a targeted agent in NSCLC cell lines. The present study co-cultured PP121 with healthy human astrocytes, a prevalent cell type located in the brain of NSCLC brain metastases. To date, few preclinical studies have examined the efficacy of PP121 as an anticancer agent, and to the best of my knowledge, no previous studies have previously evaluated its therapeutic potential in the treatment of NSCLC. To investigate the clinical heterogeneity of NSCLC, patient-derived adenocarcinoma (ADC) and squamous cell carcinoma (SCC) xenograft models were used, which exhibited epidermal growth factor receptor (EGFR) mutations and mesenchymal-epithelial transition (MET) factor amplifications. Notably, both EGFR and MET are known contributors to tyrosine kinase inhibitor resistance; thus, the aforementioned mutations and amplifications enabled the effects of PP121 to be evaluated in these solid tumors. In addition, a co-cultured model system using both NSCLC cells and astrocytes was employed to assess the effects of PP121 on the invasion of ADC and SCC cells in a multicellular environment. Results of the present study demonstrated that PP121 exerted an antitumorigenic effect in the aforementioned model systems via downregulation of pharmacodynamic targets.

Preclinical trial comparing radiotherapy alone versus standard radiochemotherapy in three human papilloma virus (HPV) negative and three HPV-positive head and neck squamous cell carcinoma (HNSCC) xenograft tumour models

PurposeTo perform a preclinical trial comparing the efficacy of fractionated radiotherapy versus radiochemotherapy with cisplatin in HPV-positive and negative human head and neck squamous cell carcinoma (HNSCC) xenografts. Material and methodsThree HPV-negative and three HPV-positive HNSCC xenografts in nude mice were randomized to radiotherapy (RT) alone or to radiochemotherapy (RCT) with weekly cisplatin. To evaluate tumour growth time, 20 Gy radiotherapy (±cisplatin) were administered in 10 fractions over 2 weeks. Dose-response curves for local tumour control were generated for RT with 30 fractions over 6 weeks to different dose levels given alone or combined with cisplatin (RCT). ResultsOne of three investigated HPV-negative and two out of three HPV-positive tumour models showed a significant increase in local tumour control after RCT compared to RT alone. Pooled analysis of the HPV-positive tumour models showed a statistically significant and substantial benefit of RCT versus RT alone, with an enhancement ratio of 1.34. Although heterogeneity in response to both RT and RCT was also observed between the different HPV-positive HNSCC, these overall were more RT and RCT sensitive than HPV-negative models. ConclusionThe impact of adding chemotherapy to fractionated radiotherapy on local control was heterogenous, both in HPV-negative and in HPV-positive tumours, calling for predictive biomarkers. RCT substantially increased local tumour control in the pooled group of all HPV-positive tumours whereas this was not found in HPV-negative tumours. Omission of chemotherapy in HPV-positive HNSCC as part of a treatment de-escalation strategy is not supported by this preclinical trial.

Rapid and Quantitative Intraoperative Pathology-Assisted Surgery by Paired-Agent Imaging-Derived Confidence Map.

In nonmetastatic head and neck cancer treatment, surgical margin status is the most important prognosticator of recurrence and patient survival. Fresh frozen sectioning (FFS) of tissue margins is the standard of care for intraoperative margin assessment. However, FFS is time intensive, and its accuracy is not consistent among institutes. Mapping the epidermal growth factor receptor (EGFR) using paired-agent imaging (PAI) has the potential to provide more consistent intraoperative margin assessment in a fraction of the time as FFS. PAI was carried out through IV injection of an anti-epidermal growth factor receptor (EGFR) affibody molecule (ABY-029, eIND 122,681) and an untargeted IRDye680LT carboxylate. Imaging was performed on 4 µm frozen sections from three oral squamous cell carcinoma xenograft mouse models (n = 24, 8 samples per cell line). The diagnostic ability and tumor contrast were compared between binding potential, targeted, and untargeted images. Confidence maps were constructed based on group histogram-derived tumor probability curves. Tumor differentiability and contrast by confidence maps were evaluated. PAI outperformed ABY-029 and IRDye 680LT alone, demonstrating the highest individual receiver operating characteristic (ROC) curve area under the curve (PAI AUC: 0.91, 0.90, and 0.79) and contrast-to-noise ratio (PAI CNR: 1, 1.1, and 0.6) for FaDu, Det 562, and A253. PAI confidence maps (PAI CM) maintain high tumor diagnostic ability (PAI CMAUC: 0.91, 0.90, and 0.79) while significantly enhancing tumor contrast (PAI CMCNR: 1.5, 1.3, and 0.8) in FaDu, Det 562, and A253. Additionally, the PAI confidence map allows avascular A253 to be differentiated from a healthy tissue with significantly higher contrast than PAI. Notably, PAI does not require additional staining and therefore significantly reduces the tumor delineation time in a 5 [Formula: see text] 5mm slice from ~ 35min to under a minute. This study demonstrated that PAI improved tumor detection in frozen sections with high diagnostic accuracy and rapid analysis times. The novel PAI confidence map improved the contrast in vascular tumors and differentiability in avascular tumors. With a larger database, the PAI confidence map promises to standardize fluorescence imaging in intraoperative pathology-assisted surgery (IPAS).

Conjugation of the Fn14 Ligand to a SMAC Mimetic Selectively Suppresses Experimental Squamous Cell Carcinoma in Mice

The mimetic of SMAC induced cell death in cancers by depleting the inhibitor of apoptosis proteins. Recent studies showed that Fn14 is overexpressed in the cells of squamous cell carcinoma (SCC), providing a promising candidate target for selective antitumor therapy. In this study, we conjugated a small-molecule SMAC mimetic MV1 to the ligand of Fn14, TWEAK. Our results showed that TWEAK‒MV1 conjugate retained adequate binding specificity to Fn14-positive SCC cells invitro and accumulated selectively in tumor tissue of cutaneous SCC xenografts mice after intraperitoneal administration. This conjugation compound exhibited remarkable effectiveness in suppressing tumor growth and extending overall survival without causing significant side effects in SCC xenograft mice. Moreover, TWEAK‒MV1 conjugate greatly enhanced both apoptotic and necroptotic cell death both invitro and invivo, accompanied by a cellular inhibitor of apoptosis proteins degradation as well as activation of receptor-interacting protein kinase. Taken together, our preclinical data suggested that the designed conjugation compound of TWEAK and MV1 might provide a potential therapeutic strategy for cutaneous SCC with improved antitumor efficacy and negligible toxicity.

A Two-In-One Nanoprodrug for Photoacoustic Imaging-Guided Enhanced Sonodynamic Therapy.

Sonodynamic therapy (SDT) is garnering considerable attention in cancer treatment due to its non-invasive nature and the potential of spatiotemporal control. However, the high level of glutathione (GSH) in cancer cells can alleviate the SDT-mediated ROS-damages, resulting in a reduced SDT effect. Here, a two-in-one nano-prodrug for photoacoustic imaging-guided enhanced SDT against skin cancers is synthesized. A dual-prodrug molecule (DOA) of sulfide dioxide (SO2 ) and 5-aminolevulinic acid (ALA) is first synthesized and then co-assembled with methoxyl poly(ethylene glycol)-b-poly(l-lysine) (mPEG-b-PLL) to generate the two-in-one prodrug nanoparticles (P-DOA NPs). The P-DOA NPs simultaneously released ALA and SO2 in response to the overexpressed GSH in tumor cells. The released ALA is metabolically converted into protoporphyrin IX (PpIX) in tumor cells for SDT and photoacoustic imaging. Meanwhile, the released SO2 , together with the consumption of GSH based on the reaction of DOA in P-DOA NPs with intracellular GSH, can significantly increase the intracellular ROS content, leading to enhanced SDT. As a result, the P-DOA NPs significantly inhibited the growth of melanoma and squamous cell carcinoma xenografts in mouse models under the guidance of real-time photoacoustic imaging. Therefore, this novel two-in-one nano-prodrug is promising for effective SDT against skin cancers.

NIR-II Navigation with an EGFR-Targeted Probe Improves Imaging Resolution and Sensitivity of Detecting Micrometastases in Esophageal Squamous Cell Carcinoma Xenograft Models.

The survival rate of esophageal squamous carcinoma (ESCC) after surgical resection is estimated to be only 30.3% due to the difficulty in identifying microinfiltration and subtle metastases. In this study, we explored the value of near-infrared fluorescence in the second window (NIR-II) using an epidermal growth factor receptor (EGFR)-targeted probe (cetuximab-IR800) for the intraoperative navigation of ESCC in xenograft mouse models. Immunohistochemical results showed that EGFR was aberrantly expressed in 94.49% (120/127) of ESCC tissues and 90.63% (58/64) of metastatic lymph nodes. Western blot results demonstrated that EGFR protein was highly expressed in ESCC cell lines. Flow cytometry data revealed that cetuximab-IR800 showed a stronger binding specificity in EGFR-positive KYSE-30 cells than in A2780 control cells (P < 0.01). In vivo imaging data showed that the ratio of mean fluorescent intensity (MFI) and tumor to background (TBR) was significantly higher in KYSE-30 subcutaneous tumors with the infusion of cetuximab-IR800 than in those with the infusion of IgG1-IR800 (P < 0.05). Surgical navigation with NIR-II imaging showed that the TBR in orthotopic ESCC was significantly higher than that of NIR in the first window (NIR-I) (2.11 ± 0.46 vs 1.58 ± 0.31, P < 0.05), and NIR-II was more sensitive than NIR-I in detecting subcentimeter metastases (94.87% (37/39) vs 58.97% (23/39), P < 0.001). In conclusion, cetuximab-IR800 with high specificity for ESCC was first used in NIR-II surgical navigation. This probe showed better imaging resolution and higher sensitivity in detecting subtle metastases derived from an orthotopic ESCC model than NIR-I, which indicates that NIR-II has promise in guiding precise surgery for ESCC patients.

Exosomes derived from stem cells of human deciduous exfoliated teeth inhibit angiogenesis in vivo and in vitro via the transfer of miR-100-5p and miR-1246

BackgroundAnti-angiogenic therapy has been shown to be a promising strategy for anti-tumor treatment. Increasing evidence indicates that tumor angiogenesis is affected by exosomes that are secreted by mesenchymal stem cells (MSCs), but whether exosomes derived from MSCs suppress or promote angiogenesis remain paradoxical. The purpose of this study focused on understanding the potential role of exosomes derived from stem cells of human deciduous exfoliated teeth (SHED-Exos) in regulating angiogenesis and the underlying molecular mechanism.MethodsExosomes were isolated from supernatants of SHED cells using an exosome purification kit and were characterized by transmission electron microscopy, nanoparticle tracking analysis and western blot analysis. Cell Counting Kit-8, flow cytometric assays, western blots, wound healing and transwell migration assays were performed to characterize the roles of SHED-Exos on cell proliferation, apoptosis and migration of human umbilical vein endothelial cells (HUVECs). The anti-angiogenic activity of SHED-Exos was assessed via a tube formation assay of endothelial cells and angiogenesis-related factors were analyzed by western blotting. In vivo, we used the chick chorioallantoic membrane (CAM) assay and an oral squamous cell carcinoma (OSCC) xenograft transplantation model with nude mice that received multi-point injections at three-day intervals to evaluate the effects on angiogenesis. Furthermore, the sequencing of microRNAs (miRNAs) in SHED-Exos was performed to investigate the underlying anti-angiogenic mechanism.ResultsThe results showed that SHED-Exos inhibit cell proliferation and migration and induce apoptosis in HUVECs. SHED-Exos suppress the tube-like structure formation of HUVECs in vitro. SHED-Exos downregulate several angiogenesis-related factors, including VEGFA, MMP-9 and ANGPT1. In vivo, the chick CAM assay verified that treatment with SHED-Exos inhibits micro-vascular formation, and importantly, significantly reduces the micro-vascular formation of tumors generated from xenografted OSCC cells, which was associated with the inhibition of tumor growth in vivo. Mechanistically, our data suggested that SHED-Exos are enriched with miR-100-5p and miR-1246 and are transferred to endothelial cells, which results in decreased tube formation via the down-regulation of VEGFA expression.ConclusionsThese results demonstrate that SHED-Exos inhibit angiogenesis in vitro and in vivo, which suggests that SHED-Exos could potentially serve as a novel and effective therapeutic approach for anti-angiogenic treatment.

Heme Sequestration Effectively Suppresses the Development and Progression of Both Lung Adenocarcinoma and Squamous Cell Carcinoma.

Lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC) are two most common subtypes of lung cancer. Here, to identify new, targetable molecular properties of both subtypes, we monitored changes in the levels of heme- and oxidative phosphorylation (OXPHOS)-related proteins during lung tumorigenesis. Heme is a central molecule for oxidative metabolism and ATP generation via OXPHOS. Notably, both lung ADC and SCC tumors can be induced in the genetically engineered KLLuc mouse model harboring the G12D Kras mutation and a conditional Lkb1 knockout. We found that the levels of the rate-limiting heme synthesis enzyme ALAS1 and uptake protein SLC48A1, along with OXPHOS complex subunits, progressively increased as lung tumorigenesis advanced. Our data demonstrated that elevated levels of heme- and OXPHOS-related proteins were associated with both ADC and SCC. Importantly, treatment of KLLuc mice with a heme-sequestering protein, HeSP2, that inhibits heme uptake in tumor cells effectively arrested lung tumor progression, and both ADC and SCC tumors were strongly suppressed. Additionally, HeSP2 effectively suppressed the growth of both SCC and ADC tumor xenografts in NOD/SCID mice. Further analyses indicated that HeSP2 effectively diminished OXPHOS in both ADC and SCC, reduced angiogenesis, alleviated tumor hypoxia, and suppressed cell proliferation. These results show that the advancing of lung tumorigenesis requires progressive increase in cellular heme synthesis and uptake, leading to intensified OXPHOS activity and ATP generation and promoting aggressive tumorigenic functions. IMPLICATIONS: Heme sequestration is an effective strategy for the suppression of both ADC and SCC tumor initiation and development.

Improved Discrimination of Tumors with Low and Heterogeneous EGFR Expression in Fluorescence-Guided Surgery Through Paired-Agent Protocols

PurposeThe goal of fluorescence-guided surgery (FGS) in oncology is to improve the surgical therapeutic index by enhancing contrast between cancerous and healthy tissues. However, optimal discrimination between these tissues is complicated by the nonspecific uptake and retention of molecular targeted agents and the variance of fluorescence signal. Paired-agent imaging (PAI) employs co-administration of an untargeted imaging agent with a molecular targeted agent, providing a normalization factor to minimize nonspecific and varied signals. The resulting measured binding potential is quantitative and equivalent to in vivo immunohistochemistry of the target protein. This study demonstrates that PAI improves the accuracy of tumor-to-healthy tissue discrimination compared to single-agent imaging for in vivo FGS.ProceduresPAI using a fluorescent anti-epidermal growth factor receptor (EGFR) affibody molecule (ABY-029, eIND 122,681) with untargeted IRDye 700DX carboxylate was compared to ABY-029 alone in an oral squamous cell carcinoma xenograft mouse model at 3 h after dye administration (n = 30).ResultsPAI significantly enhanced tumor discrimination, as compared to ABY-029 alone in low EGFR-expressing tumors and highly heterogeneous populations including multiple cell lines with varying expression (diagnostic accuracy: 0.908 vs. 0.854 and 0.908 vs. 0.822; and ROC curve AUC: 0.963 vs. 0.909 and 0.957 vs. 0.909, respectively) indicating a potential for universal FGS image thresholds to determine surgical margins. In addition, PAI achieved significantly higher diagnostic ability than ABY-029 alone 0.25–5-h post injection and exhibited a stronger correlation to EGFR expression heterogeneity.ConclusionThe quantitative receptor delineation of PAI promises to improve the surgical therapeutic index of cancer resection in a clinically relevant timeline.

Dietary Melatonin and Omega-3 Fatty Acids Induce Human Cancer Xenograft Regression In Vivo in Rats by Suppressing Linoleic Acid Uptake and Metabolism.

Melatonin, the circadian nighttime neurohormone, and eicosapentaenoic acid (EPA) and docosahexaenoic acids (DHA), which are omega-3 fatty acids (FA) found in high concentrations in fish oil (FO) and plants, abrogate the oncogenic effects of linoleic acid (LA), an omega-6 FA, on the growth of rodent tumors and human breast, prostate, and head and neck squamous cell carcinoma (HNSCC) xenografts in vivo. Here we determined and compared the long-term effects of these inhibitory agents on tumor regression and LA uptake and metabolism to the mitogenic agent 13-[S]-hydroxyoctadecadienoic acid (13-[S]-HODE) in human prostate cancer 3 (PC3) and FaDu HNSCC xenografts in tumor-bearing male nude rats. Rats in this study were split into 3 groups and fed one of 2 diets: one diet containing 5% corn oil (CO, high LA), 5% CO oil and melatonin (2 μg/mL) or an alternative diet 5% FO (low LA). Rats whose diet contained melatonin had a faster rate of regression of PC3 prostate cancer xenografts than those receiving the FO diet, while both in the melatonin and FO groups induced the same rate of regression of HNSCC xenografts. The results also demonstrated that dietary intake of melatonin or FO significantly inhibited tumor LA uptake, cAMP content, 13-[S]-HODE formation, [³H]-thymidine incorporation into tumor DNA, and tumor DNA content. Therefore, long-term ingestion of either melatonin or FO can induce regression of PC3 prostate and HNSCC xenografts via a mechanism involving the suppression of LA uptake and metabolism by the tumor cells.

Pharmacological Inhibition of HSP90 Radiosensitizes Head and Neck Squamous Cell Carcinoma Xenograft by Inhibition of DNA Damage Repair, Nucleotide Metabolism, and Radiation-Induced Tumor Vasculogenesis.

Recent preclinical studies suggest combining the HSP90 inhibitor AT13387 (Onalespib) with radiation (IR) against colon cancer and head and neck squamous cell carcinoma (HNSCC). These studies emphasized that AT13387 downregulates HSP90 client proteins involved in oncogenic signaling and DNA repair mechanisms as major drivers of enhanced radiosensitivity. Given the large array of client proteins HSP90 directs, we hypothesized that other key proteins or signaling pathways may be inhibited by AT13387 and contribute to enhanced radiosensitivity. Metabolomic analysis of HSP90 inhibition by AT13387 was conducted to identify metabolic biomarkers of radiosensitization and whether modulations of key proteins were involved in IR-induced tumor vasculogenesis, a process involved in tumor recurrence. HNSCC and non-small cell lung cancer cell lines were used to evaluate the AT13387 radiosensitization effect in vitro and in vivo. Flow cytometry, immunofluorescence, and immunoblot analysis were used to evaluate cell cycle changes and HSP90 client protein's role in DNA damage repair. Metabolic analysis was performed using liquid chromatography-Mass spectrometry. Immunohistochemical examination of resected tumors post-AT13387 and IR treatment were conducted to identify biomarkers of IR-induced tumor vasculogenesis. In agreement with recent studies, AT13387 treatment combined with IR resulted in a G2/M cell cycle arrest and inhibited DNA repair. Metabolomic profiling indicated a decrease in key metabolites in glycolysis and tricarboxylic acid cycle by AT13387, a reduction in Adenosine 5'-triphosphate levels, and rate-limiting metabolites in nucleotide metabolism, namely phosphoribosyl diphosphate and aspartate. HNSCC xenografts treated with the combination exhibited increased tumor regrowth delay, decreased tumor infiltration of CD45 and CD11b+ bone marrow-derived cells, and inhibition of HIF-1 and SDF-1 expression, thereby inhibiting IR-induced vasculogenesis. AT13387 treatment resulted in pharmacologic inhibition of cancer cell metabolism that was linked to DNA damage repair. AT13387 combined with IR inhibited IR-induced vasculogenesis, a process involved in tumor recurrence postradiotherapy. Combining AT13387 with IR warrants consideration of clinical trial assessment.