Targeting Tumor Microenvironment Akt Signaling Represents a Potential Therapeutic Strategy for Aggressive Thyroid Cancer.

Effects of the tumor microenvironment (TME) stromal cells on progression in thyroid cancer are largely unexplored. Elucidating the effects and underlying mechanisms may facilitate the development of targeting therapy for aggressive cases of this disease. In this study, we investigated the impact of TME stromal cells on cancer stem-like cells (CSCs) in patient-relevant contexts where applying in vitro assays and xenograft models uncovered contributions of TME stromal cells to thyroid cancer progression. We found that TME stromal cells can enhance CSC self-renewal and invasiveness mainly via the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. The disruption of Akt signaling could diminish the impact of TME stromal cells on CSC aggressiveness in vitro and reduce CSC tumorigenesis and metastasis in xenografts. Notably, disrupting Akt signaling did not cause detectable alterations in tumor histology and gene expression of major stromal components while it produced therapeutic benefits. In addition, using a clinical cohort, we discovered that papillary thyroid carcinomas with lymph node metastasis are more likely to have elevated Akt signaling compared with the ones without metastasis, suggesting the relevance of Akt-targeting. Overall, our results identify PI3K/Akt pathway-engaged contributions of TME stromal cells to thyroid tumor disease progression, illuminating TME Akt signaling as a therapeutic target in aggressive thyroid cancer.

Comprehensive Review: Unveiling the Pro-Oncogenic Roles of IL-1ß and PD-1/PD-L1 in NSCLC Development and Targeting Their Pathways for Clinical Management.

In the past decade, targeted therapies for solid tumors, including non-small cell lung cancer (NSCLC), have advanced significantly, offering tailored treatment options for patients. However, individuals without targetable mutations pose a clinical challenge, as they may not respond to standard treatments like immune-checkpoint inhibitors (ICIs) and novel targeted therapies. While the mechanism of action of ICIs seems promising, the lack of a robust response limits their widespread use. Although the expression levels of programmed death ligand 1 (PD-L1) on tumor cells are used to predict ICI response, identifying new biomarkers, particularly those associated with the tumor microenvironment (TME), is crucial to address this unmet need. Recently, inflammatory cytokines such as interleukin-1 beta (IL-1ß) have emerged as a key area of focus and hold significant potential implications for future clinical practice. Combinatorial approaches of IL-1ß inhibitors and ICIs may provide a potential therapeutic modality for NSCLC patients without targetable mutations. Recent advancements in our understanding of the intricate relationship between inflammation and oncogenesis, particularly involving the IL-1ß/PD-1/PD-L1 pathway, have shed light on their application in lung cancer development and clinical outcomes of patients. Targeting these pathways in cancers like NSCLC holds immense potential to revolutionize cancer treatment, particularly for patients lacking targetable genetic mutations. However, despite these promising prospects, there remain certain aspects of this pathway that require further investigation, particularly regarding treatment resistance. Therefore, the objective of this review is to delve into the role of IL-1ß in NSCLC, its participation in inflammatory pathways, and its intricate crosstalk with the PD-1/PD-L1 pathway. Additionally, we aim to explore the potential of IL-1ß as a therapeutic target for NSCLC treatment.

Bupivacaine and Lidocaine Induce Apoptosis in Osteosarcoma Tumor Cells.

BACKGROUND: Osteosarcoma is the most common type of bone cancer in adolescents. There have been no significant improvements in outcomes since chemotherapy was first introduced. Bupivacaine and lidocaine have been shown to be toxic to certain malignancies. This study evaluates the effect of these medications on two osteosarcoma cell lines. QUESTIONS/PURPOSES: (1) Does incubation of osteosarcoma cells with bupivacaine or lidocaine result in cell death? (2) Does this result from an apoptotic mechanism? (3) Is a specific apoptotic pathway implicated? METHODS: Two cell lines were chosen to account for the inherent heterogeneity of osteosarcoma. UMR-108 is a transplantable cell line that has been used in multiple studies as a primary tumor. MNNG/HOS has a high metastatic rate in vivo. Both cell lines were exposed bupivacaine (0.27, 0.54, 1.08, 2.16, 4.33 and 8.66 mM) and lidocaine (0.66, 1.33, 5.33, 10.66, 21.32 and 42.64 mM) for 24 hours, 48 hours, and 72 hours. These concentrations were determined by preliminary experiments that found the median effective dose was 1.4 mM for bupivacaine and 7.0 mM for lidocaine in both cell lines. Microculture tetrazolium and colony formation assay determined whether cell death occurred. Apoptosis induction was evaluated by phase-contrast micrographs, flow cytometry, DNA fragmentation and reactive oxygen species (ROS). The underlying pathways were analyzed by protein electrophoresis and Western blot. All testing was performed in triplicate and compared with pH-adjusted controls. Quantitative results were analyzed without blinding. RESULTS: Both medications caused cell death in a dose- and time-dependent manner. Exposure to bupivacaine for 24 hours reduced viability of UMR-108 cells by 6 ± 0.75% (95% CI 2.9 to 9.11; p = 0.01) at 1.08 mM and 89.67 ± 1.5% (95% CI 82.2 to 95.5; p < 0.001) at 2.16 mM. Under the same conditions, MNNG/HOS viability was decreased in a similar fashion. After 24 hours, the viability of UMR-108 and MNNG/HOS cells exposed to 5.33 mM of lidocaine decreased by 25.33 ± 8.3% (95% CI 2.1 to 48.49; p = 0.03) and 39.33 ± 3.19% (95% CI 30.46 to 48.21; p < 0.001), respectively, and by 90.67 ± 0.66% (95% CI 88.82 to 92.52; p < 0.001) and 81.6 ± 0.47% (95% CI 79.69 to 82.31; p < 0.001) at 10.66 mM, respectively. After 72 hours, the viability of both cell lines was further reduced. Cell death was consistent with apoptosis based on cell morphology, total number of apoptotic cells and DNA fragmentation. The percentage increase of apoptotic UMR-108 and MNNG/HOS cells confirmed by Annexin-V positivity compared with controls was 21.3 ± 2.82 (95% CI 16.25 to 26.48; p < 0.001) and 21.23 ± 3.23% (95% CI 12.2 to 30.2; p = 0.003) for bupivacaine at 1.08 mM and 25.15 ± 4.38 (95% CI 12.9 to 37.3; p = 0.004) and 9.11 ± 1.74 (95% CI 4.35 to 13.87; p = 0.006) for lidocaine at 5.33 mM. The intrinsic apoptotic pathway was involved as the expression of Bcl-2 and survivin were down-regulated, and Bax, cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase-1 were increased. ROS production increased in the UMR-108 cells but was decreased in the MNNG/HOS cells. CONCLUSION: These findings provide a basis for evaluating these medications in the in vivo setting. Studies should be performed in small animals to determine if clinically relevant doses have a similar effect in vivo. In humans, biopsies could be performed with standard doses of these medications to see if there is a difference in biopsy tract contamination on definitive resection. CLINICAL RELEVANCE: Bupivacaine and lidocaine could potentially be used for their ability to induce and enhance apoptosis in local osteosarcoma treatment. Outcome data when these medications are used routinely during osteosarcoma treatment can be evaluated compared with controls. Further small animal studies should be performed to determine if injection into the tumor, isolated limb perfusion, or other modalities of treatment are viable.

Thyroid Carcinoma: Phenotypic Features, Underlying Biology and Potential Relevance for Targeting Therapy.

Thyroid carcinoma consists a group of phenotypically heterogeneous cancers. Recent advances in biological technologies have been advancing the delineation of genetic, epigenetic, and non-genetic factors that contribute to the heterogeneities of these cancers. In this review article, we discuss new findings that are greatly improving the understanding of thyroid cancer biology and facilitating the identification of novel targets for therapeutic intervention. We review the phenotypic features of different subtypes of thyroid cancers and their underlying biology. We discuss recent discoveries in thyroid cancer heterogeneities and the critical mechanisms contributing to the heterogeneity with emphases on genetic and epigenetic factors, cancer stemness traits, and tumor microenvironments. We also discuss the potential relevance of the intratumor heterogeneity in understanding therapeutic resistance and how new findings in tumor biology can facilitate designing novel targeting therapies for thyroid cancer.

Inhibitory Effects of Indomethacin in Human MNNG/HOS Osteosarcoma Cell Line In Vitro.

Recurrence or metastasis remains the major cause of poor prognosis and mortality in Osteosarcoma patients. Therefore, development of more effective therapeutic approaches is required. We showed that indomethacin, significantly induces apoptosis in MNNG/HOS cell line, which was confirmed by morphological changes, increased Annexin-V + cells and nuclear fragmentation. Apoptosis was accompanied by increased cleavage of caspase-3 and PARP, suggesting activation of caspase-dependent cell death. Indomethacin significantly decreased the expression of ß-catenin, a key player in tumor metastasis. These results indicate that indomethacin may have the potential to be used as neoadjuvant or adjuvant treatment; however, additional studies are required.

Subpopulations of cancer stem cells found in papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is the most common form of thyroid cancer and while it has a generally good prognosis, tumor recurrence remains a major clinical challenge. Studying laboratory cell lines as well as clinical specimens indicate that PTC may follow the cancer stem cell (CSC) model. However, CSC characteristics relevant in PTC initiation and progression remain largely unknown. Here we studied a population of sphere-growing tumor cells isolated from primary cultures of clinical PTC. These sphere-growing cells consisted of aldehyde dehydrogenase positive (ALDH+) and ALDH negative (ALDH-) cell subpopulations and demonstrated a hierarchical pattern of cell division. Using combinations of selective depletion, specific inhibition and cell sorting, we found that both subpopulations of the sphere cells were able to self-renew and initiate xenograft tumors independently, and fulfilled the definition of CSC. Importantly, when the subpopulations functioned together, the cancer-initiation efficiency and the xenograft tumor progression were significantly enhanced compared to either subpopulation alone. These data revealed crucial roles of ALDH- CSC in PTC biology and suggested that CSC subpopulations function cooperatively to control PTC initiation and progression. Together, our study indicates that CSC subpopulations isolated from clinical specimens offer unprecedented opportunities for investigating PTC pathogenesis and developing effective therapies.

Immunoseroproteomic Profiling in African American Men with Prostate Cancer: Evidence for an Autoantibody Response to Glycolysis and Plasminogen-Associated Proteins.

African American (AA) men suffer from a disproportionately high incidence and mortality of prostate cancer (PCa) compared with other racial/ethnic groups. Despite these disparities, African American men are underrepresented in clinical trials and in studies on PCa biology and biomarker discovery. We used immunoseroproteomics to profile antitumor autoantibody responses in AA and European American (EA) men with PCa, and explored differences in these responses. This minimally invasive approach detects autoantibodies to tumor-associated antigens that could serve as clinical biomarkers and immunotherapeutic agents. Sera from AA and EA men with PCa were probed by immunoblotting against PC3 cell proteins, with AA sera showing stronger immunoreactivity. Mass spectrometry analysis of immunoreactive protein spots revealed that several AA sera contained autoantibodies to a number of proteins associated with both the glycolysis and plasminogen pathways, particularly to alpha-enolase (ENO1). The proteomic data is deposited in ProteomeXchange with identifier PXD003968. Analysis of sera from 340 racially diverse men by enzyme-linked immunosorbent assays (ELISA) showed higher frequency of anti-ENO1 autoantibodies in PCa sera compared with control sera. We observed differences between AA-PCa and EA-PCa patients in their immunoreactivity against ENO1. Although EA-PCa sera reacted with higher frequency against purified ENO1 in ELISA and recognized by immunoblotting the endogenous cellular ENO1 across a panel of prostate cell lines, AA-PCa sera reacted weakly against this protein by ELISA but recognized it by immunoblotting preferentially in metastatic cell lines. These race-related differences in immunoreactivity to ENO1 could not be accounted by differential autoantibody recognition of phosphoepitopes within this antigen. Proteomic analysis revealed differences in the posttranslational modification profiles of ENO1 variants differentially recognized by AA-PCa and EA-PCa sera. These intriguing results suggest the possibility of race-related differences in the antitumor autoantibody response in PCa, and have implications for defining novel biological determinants of PCa health disparities.

Proteomic Profiling of Serum-Derived Exosomes from Ethnically Diverse Prostate Cancer Patients.

Prostate cancer (PCa) remains the most frequently diagnosed male malignancy in Western countries and the second most common cause of male cancer death in the United States. The relatively elevated PCa incidence and mortality among African American men makes this cancer type a challenging health disparity disease. To increase the chance for successful trea tment, earlier detection and prediction of tumor aggress iveness will be important and need to be resolved. This study demonstrates that small membrane-bound vesicles shed from the tumor called exosomes contain ethnically and tumor-specific biomarkers, and could be exploited for their diagnostic and therapeutic potential.

Early diagnostic value of survivin and its alternative splice variants in breast cancer.

BACKGROUND: The inhibitor of apoptosis (IAP) protein Survivin and its splice variants are differentially expressed in breast cancer tissues. Our previous work showed Survivin is released from tumor cells via small membrane-bound vesicles called exosomes. We, therefore, hypothesize that analysis of serum exosomal Survivin and its splice variants may provide a novel biomarker for early diagnosis of breast cancer. METHODS: We collected sera from forty breast cancer patients and ten control patients who were disease free for 5 years after treatment. In addition, twenty-three paired breast cancer tumor tissues from those same 40 patients were analyzed for splice variants. Serum levels of Survivin were analyzed using ELISA and exosomes were isolated from this serum using the commercially available ExoQuick kit, with subsequent Western blots and immunohistochemistry performed. RESULTS: Survivin levels were significantly higher in all the breast cancer samples compared to controls (p < 0.05) with exosome amounts significantly higher in cancer patient sera compared to controls (p < 0.01). While Survivin and Survivin-∆Ex3 splice variant expression and localization was identical in serum exosomes, differential expression of Survivin-2B protein existed in the exosomes. Similarly, Survivin and Survivin-∆Ex3 proteins were the predominant forms detected in all of the breast cancer tissues evaluated in this study, whereas a more variable expression of Survivin-2B level was found at different cancer stages. CONCLUSION: In this study we show for the first time that like Survivin, the Survivin splice variants are also exosomally packaged in the breast cancer patients' sera, mimicking the survivin splice variant pattern that we also report in breast cancer tissues. Differential expression of exosomal-Survivin, particularly Survivin-2B, may serve as a diagnostic and/or prognostic marker, a "liquid biopsy" if you will, in early breast cancer patients. Furthermore, a more thorough understanding of the role of this prominent antiapoptotic pathway could lead to the development of potential therapeutics for breast cancer patients.

Regulatory and Interacting Partners of PDLIM7 in Thyroid Cancer.

Enigma protein, encoded by the PDLIM7 gene, is overexpressed in thyroid cancer in a stage-dependent manner, suggesting a potential involvement in the initiation and progression of thyroid cancer. The Enigma interacts with several cellular pathways, including PI3K/AKT, MDM2, and BMP-1. The Enigma is regulated by microRNAs. Specifically, we showed that the Enigma protein upregulation corresponds to the downregulation of Let-7 family genes. There is limited research on the interactions and regulation of the Enigma with other proteins/genes in thyroid cancer tissues, indicating a gap in current knowledge. Our aim is to establish the Enigma as a biomarker. We also aim to study the interacting partners of the Enigma signaling pathways and their probable miRNA regulation in thyroid cancer progression. Using Western blotting, densitometric analysis, immunoprecipitation (IP), and reverse IP, we detected the protein expression and protein-protein interactions in the corresponding papillary thyroid carcinomas (PTCs). Utilizing real-time qPCR assay and Pearson's correlation test, we highlighted the correlation between PDLIM7 and Let-7g gene expression in the same tissues. The results showed the differential upregulations of the Enigma protein in different stages of PTCs compared to benign tissues along with AKT, VDR, BMP-1, and MDM2 proteins. Loss of DBP was observed in a subset of PTCs. Strong interactions of the Enigma with PI3K/AKT and MDM2 were noted, along with a weaker BMP-1 interaction. Pearson's correlation coefficient analysis between PDLIM7 and let-7g gene expression was significant (p < 0.05); however, there was a weak inverse correlation (r = -0.27). The study suggests the potential utility of the PDLIM7-qPCR assay as a biomarker for thyroid cancer. The Enigma's interactions with key signaling pathways may provide valuable insights into the development of thyroid cancer. The study contributes to understanding the molecular mechanisms involving the Enigma protein in thyroid cancer and highlights its potential as a biomarker.

Vitamin D activates FBP1 to block the Warburg effect and modulate blast metabolism in acute myeloid leukemia.

Acute myeloid leukemia (AML) has the lowest survival rate among the leukemias. Targeting intracellular metabolism and energy production in leukemic cells can be a promising therapeutic strategy for AML. Recently, we presented the successful use of vitamin D (1,25VD3) gene therapy to treat AML mouse models in vivo. In this study, recognizing the importance of 1,25VD3 as one of only 2 molecules (along with glucose) photosynthesized for energy during the beginning stage of life on this planet, we explored the functional role of 1,25VD3 in AML metabolism.Transcriptome database (RNA-seq) of four different AML cell lines revealed 17,757 genes responding to 1,25VD3-treatment. Moreover, we discovered that fructose-bisphosphatase 1 (FBP1) noticeably stands out as the only gene (out of 17,757 genes) with a 250-fold increase in gene expression, which is known to encode the key rate-limiting gluconeogenic enzyme fructose-1,6-bisphosphatase. The significant increased expression of FBP1 gene and proteins induced by 1,25VD3 was confirmed by qPCR, western blot, flow cytometry, immunocytochemistry and functional lactate assay. Additionally, 1,25VD3 was found to regulate different AML metabolic processes including gluconeogenesis, glycolysis, TCA, de novo nucleotide synthesis, etc. In summary, we provided the first evidence that 1,25 VD3-induced FBP1 overexpression might be a novel therapeutic target to block the "Warburg Effect" to reduce energy production in AML blasts.

Prior exposure to an attenuated Listeria vaccine does not reduce immunogenicity: pre-clinical assessment of the efficacy of a Listeria vaccine in the induction of immune responses against HIV.

BACKGROUND: We have evaluated an attenuated Listeria monocytogenes (Lm) candidate vaccine vector in nonhuman primates using a delivery regimen relying solely on oral vaccination. We sought to determine the impact of prior Lm vector exposure on the development of new immune responses against HIV antigens. FINDINGS: Two groups of rhesus macaques one Lm naive, the other having documented prior Lm vector exposures, were evaluated in response to oral inoculations of the same vector expressing recombinant HIV-1 Gag protein. The efficacy of the Lm vector was determined by ELISA to assess the generation of anti-Listerial antibodies; cellular responses were measured by HIV-Gag specific ELISpot assay. Our results show that prior Lm exposures did not diminish the generation of de novo cellular responses against HIV, as compared to Listeria-naïve monkeys. Moreover, empty vector exposures did not elicit potent antibody responses, consistent with the intracellular nature of Lm. CONCLUSIONS: The present study demonstrates in a pre-clinical vaccine model, that prior oral immunization with an empty Lm vector does not diminish immunogenicity to Lm-expressed HIV genes. This work underscores the need for the continued development of attenuated Lm as an orally deliverable vaccine.

A rare case of extremely delayed osseous metastasis of pineoblastoma.

Pineoblastoma is a rare, primitive, and malignant tumor arising from the parenchyma of the pineal gland. It typically metastasizes along the cerebral neural axis, with rare extraneural metastasis and even more rare intraosseous extraneural metastasis. A patient with pineoblastoma, initially treated with chemotherapy, presented 10 years after initial diagnosis with multiple osseous metastases including his pelvis, femur, and vertebrae, and is currently undergoing chemotherapy.

A case report of imiquimod topical therapy as treatment for cutaneous metastasis of breast cancer.

Cutaneous metastasis of breast cancer carries a poor prognosis, invokes a poor quality of life, and increases mortality by raising one's risk of bleeding and infection. Currently, options for treatment are systemic chemotherapy, surgical resection and radiation. These treatments are invasive and can have toxic side effects. A 50-year-old African-American woman with stage IV breast cancer with cutaneous metastasis to the left anterior chest and left supraclavicular area was successfully treated with topical imiquimod. She experienced improvement in appearance and symptoms within several months of starting treatment, resulting in near resolution of her cutaneous metastasis. Imiquimod is currently approved for several cutaneous conditions and has the potential to treat cutaneous metastasis of breast cancer.

Let-7i Reduces Aggressive Phenotype and Induces BRCAness in Ovarian Cancer Cells.

High-grade serous carcinoma of the ovary is a deadly gynecological cancer with poor long-term survival. Dysregulation of microRNAs has been shown to contribute to the formation of cancer stem cells (CSCs), an important part of oncogenesis and tumor progression. The let-7 family of microRNAs has previously been shown to regulate stemness and has tumor suppressive actions in a variety of cancers, including ovarian. Here, we demonstrate tumor suppressor actions of let-7i: repression of cancer cell stemness, inhibition of migration and invasion, and promotion of apoptosis, features important for cancer progression, relapse, and metastasis. Let-7i over-expression results in increased sensitivity to the PARP inhibitor olaparib in samples without BRCA mutations, consistent with induction of BRCAness phenotype. We also show that let-7i inhibits the expression of several factors involved in the homologous recombination repair (HRR) pathway, providing potential mechanisms by which the BRCAness phenotype could be induced. These actions of let-7i add to the rationale for use of this miRNA as a treatment for ovarian cancer patients, including those without mutations in the HRR pathway.

Ex vivo isolation, expansion and bioengineering of CCR7+CD95-/or CD62L+CD45RA+ tumor infiltrating lymphocytes from acute myeloid leukemia patients' bone marrow.

T cell based immunotherapies can be applicable to acute myeloid leukemia (AML). Therefore, the selection of optimal T cells, cell manufacturing, and therapeutic T cell engineering are essential for the development of effective adoptive T cell therapies for AML. Autologous tumor-infiltrating lymphocytes (TILs) have been in clinical trials to treat solid malignancies. Herein, we assessed whether TILs can be isolated from the bone marrow (BM) of AML patients, expanded ex vivo and utilized as a novel therapeutic strategy for AML. To this end, firstly we analyzed the immunophenotypes of a series of primary BM samples from AML patients (N = 10) by flow cytometry. We observed a variable amount of CD3+ TILs (range ∼2.3-∼32.6% of mononuclear cells) among BM samples. We then developed a novel protocol that produced a three-log ex vivo expansion of TILs isolated from AML patient BM (N = 10) and peripheral blood (PB) (N = 10), including from patients with a low number of CD3+ T cells, within 3, 4 weeks. Further, we identified previously described naïve T cells (CCR7+CD95-/or CD62L+CD45RA+) in AML BM and PB samples, which seemed to be required for a successful TILs ex vivo expansion. Finally, we showed that the expanded TILs could: (1) cause cytotoxicity to autologous AML blasts ex vivo (90.6% in control without T cell treatment vs. 1.89% in experimental groups with PB derived T cells and 1.77% in experimental groups with BM derived TILs, p < 0.01), (2) be genetically engineered to express CYP27B1 gene, and (3) infiltrate the BM and reside in close proximity to pre-injected autologous AML blasts of engrafted immunodeficiency mice. Altogether, these results provide a rationale for further studies of the therapeutic use of TILs in AML.

Suboptimal engagement of the T-cell receptor by a variety of peptide-MHC ligands triggers T-cell anergy.

T cells recognize antigen via the T-cell receptor (TCR) and produce a spectrum of responses that range from activation to anergy or cell death. The variety of outcomes may be dictated by the strength of the signals transmitted upon cognate recognition of the TCR. The physiological outcome of TCR engagement is determined by several factors, including the avidity of the ligand for TCR, the duration of engagement, and the presence and nature of accessory molecules present on antigen-presenting cells (APCs). In this review, we discuss a model of anergy induced by presentation of low densities of peptide-major histocompatibility complex (MHC) ligand in CD4(+) T cells and compare it to anergy induced by altered peptide ligands in an effort to identify a unifying mechanism. We suggest that altered peptide ligand (APL) and low densities of agonist ligands induce anergy by engaging less than optimal numbers of TCRs. The physiological impacts of anergy in memory CD4(+) T cells are discussed.

Anergy in memory CD4+ T cells is induced by B cells.

Induction of tolerance in memory T cells has profound implications in the treatment of autoimmune diseases and transplant rejection. Previously, we reported that the presentation of low densities of agonist peptide/MHC class II complexes induced anergy in memory CD4(+) T cells. In the present study, we address the specific interaction of different types of APCs with memory CD4(+) T cells. A novel ex vivo anergy assay first suggested that B cells induce anergy in memory T cells, and an in vivo cell transfer assay further confirmed those observations. We demonstrated that B cells pulsed with defined doses of Ag anergize memory CD4 cells in vivo. We established that CD11c(+) dendritic cells do not contribute to anergy induction to CD4 memory T cells, because diphtheria toxin receptor-transgenic mice that were conditionally depleted of dendritic cells optimally induced anergy in memory CD4(+) T cells. Moreover, B cell-deficient muMT mice did not induce anergy in memory T cells. We showed that B2 follicular B cells are the specific subpopulation of B cells that render memory T cells anergic. Furthermore, we present data showing that anergy in this system is mediated by CTLA-4 up-regulation on T cells. This is the first study to demonstrate formally that B cells are the APCs that induce anergy in memory CD4(+) T cells.

Antiproliferative effect of bupivacaine on patient-derived sarcoma cells.

Sarcomas are rare tumors with limited treatment options. Although chemotherapy is standard for certain subtypes, overall survival has not improved in several decades. Bupivacaine has been shown to induce apoptosis and prevent cell growth in multiple different types of malignancies but has not been studied in sarcoma. The current study evaluated the effects of bupivacaine on multiple patient-derived sarcoma cells and a commercial sarcoma cell line. Multiple patient-derived sarcoma cell subtypes and a commercial synovial cell sarcoma cell line were exposed to bupivacaine for different durations and at different concentrations. The patient-derived cells included a high-grade conventional osteosarcoma, a high-grade undifferentiated pleomorphic sarcoma of bone, and a high-grade synovial sarcoma. Flow cytometry and an MTT assay were used to evaluate whether a treatment effect was observed. Treatment of all the subtypes of sarcomas in this study with bupivacaine demonstrated a time- and dose-dependent increase in apoptosis and decrease in cell viability. A cell viability assay demonstrated that the IC50 was between 0.04 and 0.05% and that the treatment effect occurred at clinically relevant doses in vitro. Bupivacaine was toxic to both the patient-derived cells and the commercial cell line at doses commonly used in the clinical setting. These findings provide a foundation for further in vivo studies to evaluate whether these effects will translate to the clinical setting. Although further research is necessary, bupivacaine shows promise as not only an adjunct for pain management but as a treatment modality for sarcoma.

Epithelial/mesenchymal heterogeneity of high-grade serous ovarian carcinoma samples correlates with miRNA let-7 levels and predicts tumor growth and metastasis.

Patient-derived samples present an advantage over current cell line models of high-grade serous ovarian cancer (HGSOC) that are not always reliable and phenotypically faithful models of in vivo HGSOC. To improve upon cell line models of HGSOC, we set out to characterize a panel of patient-derived cells and determine their epithelial and mesenchymal characteristics. We analyzed RNA and protein expression levels in patient-derived xenograft (PDX) models of HGSOC, and functionally characterized these models using flow cytometry, wound healing assays, invasion assays, and spheroid cultures. Besides in vitro work, we also evaluated the growth characteristics of PDX in vivo (orthotopic PDX). We found that all samples had hybrid characteristics, covering a spectrum from an epithelial-to-mesenchymal state. Samples with a stronger epithelial phenotype were more active in self-renewal assays and more tumorigenic in orthotopic xenograft models as compared to samples with a stronger mesenchymal phenotype, which were more migratory and invasive. Additionally, we observed an inverse association between microRNA let-7 (lethal-7) expression and stemness, consistent with the loss of let-7 being an important component of the cancer stem cell phenotype. We observed that lower let-7 levels were associated with the epithelial state and a lower epithelial mesenchymal transition (EMT) score, more efficient spheroid and tumor formation, and increased sensitivity to platinum-based chemotherapy. Surprisingly, in these HGSOC cells, stemness could be dissociated from invasiveness: Cells with lower let-7 levels were more tumorigenic, but less migratory, and with a lower EMT score, than those with higher let-7 levels. We conclude that let-7 expression and epithelial/mesenchymal state are valuable predictors of HGSOC proliferation, in vitro self-renewal, and tumor burden in vivo.