Low-grade Tumor Cells Research Articles

Breast cancer cell-derived extracellular vesicles promote CD8+ T cell exhaustion via TGF-β type II receptor signaling

Cancer immunotherapies have shown clinical success in various types of tumors but the patient response rate is low, particularly in breast cancer. Here we report that malignant breast cancer cells can transfer active TGF-β type II receptor (TβRII) via tumor-derived extracellular vesicles (TEV) and thereby stimulate TGF-β signaling in recipient cells. Up-take of extracellular vesicle-TβRII (EV-TβRII) in low-grade tumor cells initiates epithelial-to-mesenchymal transition (EMT), thus reinforcing cancer stemness and increasing metastasis in intracardial xenograft and orthotopic transplantation models. EV-TβRII delivered as cargo to CD8+ T cells induces the activation of SMAD3 which we demonstrated to associate and cooperate with TCF1 transcription factor to impose CD8+ T cell exhaustion, resulting in failure of immunotherapy. The levels of TβRII+ circulating extracellular vesicles (crEV) appears to correlate with tumor burden, metastasis and patient survival, thereby serve as a non-invasive screening tool to detect malignant breast tumor stages. Thus, our findings not only identify a possible mechanism by which breast cancer cells can promote T cell exhaustion and dampen host anti-tumor immunity, but may also identify a target for immune therapy against the most devastating breast tumors.

Inside Front Cover: AKT1/HK2 Axis‐mediated Glucose Metabolism: A Novel Therapeutic Target of Sulforaphane in Bladder Cancer

Mol. Nutr. Food Res. 2022, 66, 202100738 DOI: 10.1002/mnfr.202100738 A unique characteristic of glucose transport aberrant-independent aerobic glycolysis exists in bladder cancer patients, and the lower grade malignant tissues have the more obvious abnormality. Sulforaphane, an isothiocyanate derived from glucosinolates in cruciferous vegetables, has been proven to inhibit glycolysis and mitochondrial oxidative phosphorylation via blocking of the AKT1/HK2 axis and PDH expression, especially in low grade tumor cells. This is reported by Minghua Ren, Yujuan Shan, and co-workers in article number 2100738.

Single-cell analysis of copy-number alterations in serous ovarian cancer reveals substantial heterogeneity in both low- and high-grade tumors

ABSTRACT Unusually high aneuploidy is a hallmark of epithelial serous ovarian cancer (SOC). Previous analyses have focused on aneuploidy on average across all tumor cells. With the expansion of single-cell sequencing technologies, however, an analysis of copy number heterogeneity cell-to-cell is now technically feasible. Here, we describe an analysis of single-cell RNA sequencing (scRNA-seq) data to infer arm-level aneuploidy in individual serous ovarian cancer cells. By first clustering high-quality sequenced epithelial versus non-epithelial cells, high-confidence tumor cell populations were identified. InferCNV was used to predict segmented copy-number alterations (CNAs), which were then used to determine arm-level aneuploidy at the single-cell level. Control comparisons of normal cells to normal cells showed zero arm-level aneuploidy, whereas a median of four aneuploid events were detectable in cancer cells. A heterogeneity analysis of high-grade tumor cells compared to low-grade tumor cells showed similar levels of cell-to-cell variation between cancer grades. Metastatic tumors potentially showed selection pressure with reduced cell-to-cell variation compared to cells from primary tumors. Minor cell populations with CNAs similar to metastatic cells were identified within the matched primary tumors. Taken together, these results provide a minimum estimate for single-cell aneuploidy in serous ovarian cancer and demonstrate the utility of single-cell sequencing for CNA analysis.

GSK‑3 inhibitor CHIR99021 enriches glioma stem‑like cells.

Glioblastoma (GBM) is the most prevalent and lethal primary intrinsic brain cancer. The disease is essentially incurable, with glioblastomas characterized by resistance to both chemotherapy and radiotherapy, as well as by rapid tumor progression, all of which are mainly ascribed to glioma stem-like cells (GSLCs). In the present study, an improved model that is more similar to clinical GBM was constructed. Twenty clinical glioma samples were collected to obtain primary low-grade tumor cells. The cells were either maintained in serum-free medium as primary glioma-based cells (PGBCs) or cultured in the same medium with CHIR99021 as GSLCs. Then, the molecular and ultrastructural differences between the two cell groups were determined. Furthermore, the proliferation and migration of the GSLCs were examined and the potential mechanisms were investigated. Finally, temozolomide resistance in vitro and in the mouse model was assessed to study the properties of the induced GSLCs. The primary low-grade tumor cells extracted from surgical samples were enriched with GSLC properties, with high expression levels of CD133 and Nestin in 100 nM CHIR99021. The GSLCs exhibited high proliferation and migration. Furthermore, the expression of the PI3K/AKT signaling pathway and that of related genes and proteins were significantly enhanced by CHIR99021. The animal study also revealed high levels of STAT3, mTOR, NF-κB, and VEGF in the GSLC-transplanted mice. CHIR99021 could stably enhance GSLC properties in patient-derived glioma samples. It may provide a useful model for further study, helping to understand the pathogenesis of therapeutic resistance and to screen drug candidates.

Abstract LB-202: Role of the FOXA transcription factors in shaping distinct transcriptional programs in human pancreatic cancers

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest types of cancer and is characterized by a high intra-tumor heterogeneity, with the coexistence of well- and poorly-differentiated cells in virtually all the tumor cases. FOXA1/2 play a fundamental role in pancreas development and are believed to act as pioneer factors, namely to create accessible chromatin by displacing nucleosomes early in development and making genomic regulatory elements available to other transcriptional regulators. Therefore, we asked whether FOXA1/2 also control differentiation in human PDAC. Whereas FOXA1 showed a remarkable propensity to be expressed selectively by well-differentiated tumor areas and cell lines, FOXA2 showed a broad profile of expression throughout PDAC stages. Through the combination of genomic and functional assays we found that FOXA2 contributed to activate distinct transcriptional programs in tumor cells of different grades. Such complexity in FOXA2 activity specifically depended on its interaction with distinct transcription factors selectively expressed in high- or low-grade tumor cells. While in low-grade (well-differentiated) PDAC cells FOXA2 binding specificity relied on the interaction with the epithelial transcription factor HNF1beta, in high-grade cells it depended on the cooperation with HOXB8 and HOXB9. Knocking out these transcription factors was sufficient to abolish differential recruitment of FOXA2 to grade specific genomic sites. In summary, we found that FOXA2 is broadly express throughout PDAC stages and, through the interaction with different transcription factors, shapes the transcriptional program of the different tumor grades in pancreatic cancer. These findings help to shed light on the complexity of PDAC heterogeneity and on the circuitries that maintain PDAC differentiation and grading. Citation Format: Marta Milan, Gioacchino Natoli. Role of the FOXA transcription factors in shaping distinct transcriptional programs in human pancreatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-202.

Breast carcinoma with osteoclast-like giant cells: A cytological-pathological correlation with a literature review.

Breast carcinoma with osteoclast-like giant cells (OGCs) is a rare disease characterized by the infiltration of OGCs in the tumor; however, cytological aspects of this tumor type remain elusive. We examined the cytological features in fine needle aspiration (FNA) biopsy smears obtained from 5 patients who were histologically diagnosed with breast carcinoma with OGCs. We compared FNA and clinicopathological findings with results from the published literature. Histological assessment of the resected samples showed that all tumors exhibited a histological grade 1 phenotype with a predominant cribriform architecture. Four patients were estrogen receptor positive, and 1 patient showed a triple negative phenotype. All patients survived without tumor recurrence. In the FNA smears, tumor cells were arranged in loosely cohesive clusters, characterized by varying degrees of OGCs infiltration and rare formation of solid tumor nests. Occasionally, 2- or 3-dimensional clusters of tumor cells were found, accompanied by OGCs at the peripheral regions. In all patients, tumor cells were small without severe nuclear atypia. None of the patients showed significant background necrosis. In summary, cytological features of breast carcinoma with OGCs are characterized by loose aggregates of low grade tumor cells, the presence of OGCs, and the absence of necrosis, all of which were consistent with features reported previously. This peculiar form of breast tumors should be included in the differential diagnosis, when physicians encounter FNA findings including low grade ductal carcinoma with the admixture of multinucleated giant cells or OGCs.

Concomitant multifocal gastrinomas and adenocarcinoma in the stomach: A case report and literature review

The stomach is an uncommon location for gastrinomas. Here we report a rare case of multifocal gastrinomas in the antrum with concomitant gastric adenocarcinoma and neuroendocrine cell hyperplasia. The patient was a 35-year-old man who presented with mild upper abdominal discomfort for 3months. Serum gastrin level was elevated to 542pg/mL (normal: <100pg/mL). Endoscopic examination revealed multiple polyps in the antrum, which proved to be carcinoid tumors on biopsy. Partial gastrectomy was performed. The surgical specimen contained multiple pedunculated polyps measuring up to 2cm in greatest dimension. Additionally, many small, sessile polyps with central umbilication were identified in the antrum and the pylorus. Histological and immunohistochemical studies of the polyps showed nests of low-grade tumor cells that were positive for gastrin, confirming a diagnosis of multifocal gastrinoma. Incidentally, one large polyp was found to harbor a focus of poorly-differentiated adenocarcinoma with signet ring cell features. Diffuse enterochromaffin-like cell hyperplasia and small carcinoid tumors were identified in the body of the stomach. Regional lymph nodes were positive for both adenocarcinoma and gastrinoma. In addition, Helicobacter pylori-like organisms were detected in the antral mucosa. The post-operative course was uneventful, and the patient's serum gastrin level dropped to 30pg/mL. He is currently receiving chemotherapy for the adenocarcinoma. This is a rare case of multifocal gastrinoma in an unusual location. It has already been established that hypergastrinemia plays a role in the pathogenesis of gastric adenocarcinoma and neuroendocrine neoplasms. Therefore, it is important to rule out these concomitant lesions, both clinically and pathologically, when dealing with a proven gastrinoma.

High expression of spindle assembly checkpoint proteins CDC20 and MAD2 is associated with poor prognosis in urothelial bladder cancer

Aneuploidy is a result of the abnormal expression of spindle assembly checkpoint (SAC) proteins and resulting abnormal spindle function during mitosis. High expression of cell division cycle 20 homolog (CDC20) and mitotic arrest defective protein 2 (MAD2), key components of the SAC, has been reported in various carcinomas. However, the clinicopathological significance of CDC20 and MAD2 expressions in urothelial carcinoma of the human bladder (UCB) is unknown. We therefore studied the expression of CDC20 and MAD2 in UCB specimens by immunohistochemistry. High expression of CDC20 and MAD2 was observed in 59.0 % (200/339) and 51.0 % (173/339) of UCB cases, respectively. Most high-grade tumor cells exhibited diffuse nuclear and/or cytoplasmic staining for CDC20 and MAD2, whereas most low-grade tumor cells and normal urothelial cells were not stained. CDC20 overexpression was associated with advanced age (p = 0.010), high grade (p < 0.001), advanced stage (p < 0.001), non-papillary growth pattern (p < 0.001), and distant metastasis (p = 0.042). Similarly, high MAD2 expression correlated with high grade (p < 0.001), advanced stage (p < 0.001), and non-papillary growth pattern (p < 0.001). In univariate survival analyses, high CDC20 expression correlated with shorter recurrence-free survival (RFS) (p = 0.032) and poorer overall survival (OS) (p = 0.007) in patients with UCB, whereas high MAD2 expression was associated with poorer OS (p = 0.008). In multivariate analyses, high CDC20 expression correlated with shorter RFS of patients with Ta stage UCB (hazard ratio, 1.91; p = 0.01). In conclusion, increased expression of CDC20 and MAD2 is related to poor prognosis of UCB.

Association of Epithelial Na+ channel (ENaC) and Acid‐Sensitive Ion Channel (ASIC) Subunits with Lipid Rafts in High Grade Gliomas

Malignant gliomas, the most common type of primary tumors of the central nervous system, are divided into four grades based on their degree of malignancy. Glioblastoma multiforme (GBM), Grade IV, is the most frequently occurring, most invasive, and has the worst prognostic outcome. Studies in our laboratory have established that high grade glioma cells express subunits of the Epithelial Na+ channel/Degenerin family, and that these cells also exhibit a basally active cation current which is not present in low grade tumor cells or normal astrocytes. The goal of our research is to characterize the pathways that regulate trafficking of this novel channel complex in gliomas by testing the hypothesis that this heteromeric channel complex is recruited to and assembled on lipid rafts prior to incorporation into the plasma membrane. By utilizing biochemical and biophysical methods, we have obtained data showing an association of the lipid raft markers caveolin‐1 and flottilin‐1 with ENaC and ASIC subunits. Cholesterol depletion experiments indicate decreased expression of these subunits in lipid raft fractions isolated from glioma cells. Overall, the results generated from these studies will provide important clues as to the physiological role of this current pathway in the biology of brain tumors. Research supported by NIH‐5T32DK7545‐23.

Advances in optical biopsy – correlation of malignancy and cell density of primary brain tumors using Raman microspectroscopic imaging

Raman spectroscopy is a promising tool towards biopsy under vision as it provides label-free image contrast based on intrinsic vibrational spectroscopic fingerprints of the specimen. The current study applied the spectral unmixing algorithm vertex component analysis (VCA) to probe cell density and cell nuclei in Raman images of primary brain tumor tissue sections. Six Raman images were collected at 785 nm excitation that consisted of 61 by 61 spectra at a step size of 2 micrometers. After data acquisition the samples were stained with hematoxylin and eosin for comparison. VCA abundance plots coincided well with histopathological findings. Raman spectra of high grade tumor cells were found to contain more intense spectral contributions of nucleic acids than those of low grade tumor cells. Similarly, VCA endmember signatures of Raman images from high grade gliomas showed increased nucleic acid bands. Further abundance plots and endmember spectra were assigned to tissue containing proteins and lipids, and cholesterol microcrystals. Since no sample preparation is required, an important advantage of the Raman imaging methodology is that all tissue components can be observed - even those that may be lost in sample staining steps. The results demonstrate how morphology and chemical composition obtained by Raman imaging correlate with histopathology and provide complementary, diagnostically relevant information at the cellular level.

Participation of the Chaperone Hsc70 in the Trafficking and Functional Expression of ASIC2 in Glioma Cells

High-grade glioma cells express subunits of the ENaC/Deg superfamily, including members of ASIC subfamily. Our previous work has shown that glioma cells exhibit a basally active cation current, which is not present in low-grade tumor cells or normal astrocytes, and that can be blocked by amiloride. When ASIC2 is present within the channel complex in the plasma membrane, the channel is rendered non-functional because of inherent negative effectors that require ASIC2. We have previously shown that high-grade glioma cells functionally express this current because of the lack of ASIC2 in the plasma membrane. We now hypothesize that ASIC2 trafficking in glioma cells is regulated by a specific chaperone protein, namely Hsc70. Our results demonstrated that Hsc70 co-immunoprecipitates with ASIC2 and that it is overexpressed in glioma cells as compared with normal astrocytes. In contrast, there was no difference in the expression of calnexin, which also co-immunoprecipitates with ASIC2. In addition, glycerol and sodium 4-phenylbutyrate reduced the amount of Hsc70 expressed in glioma cells to levels found in normal astrocytes. Transfection of Hsc70 siRNA inhibited the constitutively activated amiloride-sensitive current, decreased migration, and increased ASIC2 surface expression in glioma cells. These results support an association between Hsc70 and ASIC2 that may underlie the increased retention of ASIC2 in the endoplasmic reticulum of glioma cells. The data also suggest that decreasing Hsc70 expression promotes reversion of a high-grade glioma cell to a more normal astrocytic phenotype.

Surface Expression of ASIC2 Inhibits the Amiloride-sensitive Current and Migration of Glioma Cells

Gliomas are primary brain tumors with a complex biology characterized by antigenic and genomic heterogeneity and a propensity for invasion into normal brain tissue. High grade glioma cells possess a voltage-independent, amiloride-inhibitable, inward Na+ current. This current does not exist in normal astrocytes or low grade tumor cells. Inhibition of this conductance decreases glioma growth and cell migration making it a potential therapeutic target. Our previous results have shown that the acid-sensing ion channels (ASICs), members of the epithelial Na+ channel (ENaC)/degenerin (DEG) family of ion channels are part of this current pathway. We hypothesized that one member of the ENaC/DEG family, ASIC2, is retained intracellularly and that it is the lack of functional expression of ASIC2 at the cell surface that results in hyperactivity of this conductance in high grade gliomas. In this study we show that the chemical chaperone, glycerol, and the transcriptional regulator, sodium 4-phenylbutyrate, inhibit the constitutively activated inward current and reduce cell growth and migration in glioblastoma multiforme. The results suggest that these compounds induce the movement of ASIC2 to the plasma membrane, and once there, the basally active inward current characteristic of glioma cells is abolished by inherent negative regulatory mechanisms. This in turn compromises the ability of the glioma cell to migrate and proliferate. These results support the hypothesis that the conductance pathway in high grade glioma cells is comprised of ENaC/DEG subunits and that abolishing this channel activity promotes a reversion of a high grade glioma cell to a phenotype resembling that of normal astrocytes.

Cellular localization of acid sensing ion channels in human astrocytes and gliomas

Gliomas are primary brain tumors with a complex biology characterized by antigenic and genomic heterogeneity, and a propensity for invasion into normal brain tissue. High grade glioma cells possess a voltage-independent, amiloride-inhibitable, inward Na+ current. This current is not seen in normal astrocytes or low grade tumor cells. Inhibition of this conductance decreases glioma growth and cell migration making it a potential therapeutic target. Our results suggest that the acid sensing ion channels (ASICs), members of the Epithelial Na+ Channel (ENaC)/Degenerin (Deg) family of ion channels constitute this current pathway. We hypothesized that one member of the ENaC/Deg family, the ASIC2, is retained intracellularly, and that it is the lack of functional expression of ASIC2 at the cell surface that results in hyperactivity of this conductance in high-grade gliomas. We now show that the chemical chaperone glycerol and sodium 4-phenylbutyrate inhibit the constitutively activated inward current and reduce cell growth and migration. The results suggest that these compounds induce the movement of ASIC2 to the plasma membrane and, once there, the basally active inward current characteristic of glioma cells is abolished by inherent negative regulatory mechanisms. This, in turn, compromises the ability of the glioma cell to migrate and proliferate. These results support the hypothesis that the conductance pathway in high grade glioma cells is comprised of ENaC/Deg subunits, and that abolishing this channel activity promotes a reversion of a high grade glioma cell to a more normal astrocyte phenotype. Supported by NIH Grant CA101952 and P50CA97247.

MUCOSA-ASSOCIATED LYMPHOID TISSUE LYMPHOMA WITH INTRAOCULAR INVOLVEMENT

To report the clinicopathologic features of a patient with mucosa-associated lymphoid tissue (MALT) lymphoma of the conjunctiva and associated intraocular involvement. This study is a retrospective clinicopathologic correlative case report summarizing the clinical, radiologic, and histopathologic findings of a patient with conjunctival MALT lymphoma and associated intraocular involvement. Ophthalmic examination and fluorescein angiography demonstrated progressive conjunctival infiltration bilaterally, marked uveal effusions in the left eye, and cellular white infiltrates of the choroid in the right eye. MRI of the orbit revealed a diffusely infiltrating intra- and extraocular lesion extending around the globe and optic nerve in the left eye without evidence of intracranial extension. Conjunctival biopsy showed low-grade tumor cells, consistent with the diagnosis of MALT lymphoma. The patient was successfully treated with external beam radiation with marked clinical improvement. Conjunctival MALT lymphomas, typically indolent and localized tumors, may extend into the orbit and invade local tissues such as the choroid with devastating consequences. A conjunctival biopsy can provide an easy diagnosis of this treatable entity.

Acid-sensing Ion Channels in Malignant Gliomas

High grade glioma cells derived from patient biopsies express an amiloride-sensitive sodium conductance that has properties attributed to the human brain sodium channel family, also known as acid-sensing ion channels (ASICs). This amiloride-sensitive conductance was not detected in cells obtained from normal brain tissue or low grade or benign tumors. Differential gene profiling data showed that ASIC1 and ASIC2 mRNA were present in normal and low grade tumor cells. Although ASIC1 was present in all of the high grade glial cells examined, ASIC2 mRNA was detected in less than half. The main purpose of our work was to examine the molecular mechanisms that may underlie the constitutively activated sodium currents present in high grade glioma cells. Our results show that 1) gain-of-function mutations of ASIC1 were not present in a number of freshly resected and cultured high grade gliomas, 2) syntaxin 1A inhibited ASIC currents only when ASIC1 and ASIC2 were co-expressed, and 3) the inhibition of ASIC currents by syntaxin 1A had an absolute requirement for either gamma- or delta-hENaC. Transfection of cultured cells originally derived from high grade gliomas (U87-MG and SK-MG1) with ASIC2 abolished basal amiloride-sensitive sodium conductance; this inhibition was reversed by dialysis of the cell interior with Munc-18, a syntaxin-binding protein that typically blocks the interaction of syntaxin with other proteins. Thus, syntaxin 1A cannot inhibit Na(+) permeability in the absence of adequate plasma membrane ASIC2 expression, accounting for the observed functional expression of amiloride-sensitive currents in high grade glioma cells.

ARTIFICIAL LIVER SUPPORT DEVICES FOR FULMINANT LIVER FAILURE

Artificial liver-support devices attempt to bridge patients with fulminant hepatic failure until either a suitable liver allograft is obtained for transplantation or the patient's own liver regenerates sufficiently to resume normal function. It is thought that toxins contribute to the clinical picture of fulminant hepatic failure. The earliest reports of successful toxin removal were blood- and plasma-exchange transfusions. Given these successful case reports, mechanical liver-support devices were designed to filter toxins. These mechanical devices used hemodialysis, charcoal hemoperfusion, hemoperfusion through cation-exchange resins, hemodiabsorption, and combinations of all of these techniques as in the MARS liver-support device. Despite promising case reports and small series, no controlled studies of mechanical devices have ever showed a long-term survival benefit. Thus, the removal of presumed toxins seems to be insufficient to support patients with fulminant hepatic failure, and the biologic function of the liver must also be replaced. Attempts at replacing the biologic function have included extracorporeal liver perfusion, cross-circulation, and hepatocyte transplantation. Current technologies have combined mechanical and biologic support systems in hybrid liver-support devices. The mechanical component of these hybrid devices serves both to remove toxins and to create a barrier between the patient's serum and the biologic component of the liver-support device. The biologic component of these hybrid liver support devices may consist of liver slices, granulated liver, or hepatocytes from low-grade tumor cells or porcine hepatocytes. These biologic components are housed within bioreactors. Currently the most clinically studied bioreactors are those that use capillary hollow-fiber systems. Both the bioartificial liver by Demetrious and the extracorporeal liver-assist device by Sussman and Kelly are in clinical trials. Although the trials seemed to have yielded good survival data when the devices are used as a bridge to transplantation, the type and degree of liver support provided by these devices remains uncertain. Thus, despite decades of great progress in the field of artificial liver support, no one technique alone yet provides sufficient liver support. A hybrid system seems to be the best option at present. Still to be determined is the best tissue to use, how much liver tissue should be used, and the optimal design of the bioreactor.

Bladder cancer risk assessment with quantitative fluorescence image analysis of tumor markers in exfoliated bladder cells

The detection of potentially highly curable low-grade bladder cancers by noninvasive techniques remains an unsolved problem. Conventional cytology detects such tumors with 50% sensitivity, and addition of DNA measurements to cytology only improves sensitivity incrementally. Tumor-associated antigens potentially offer an additional diagnostic marker. In this study, the M344 antibody against a tumor-associated antigen expressed mainly by low-grade tumor cells was tested for its sensitivity and specificity, alone and in combination with DNA ploidy and cytology. Voided urine samples from 69 asymptomatic control subjects, urines and bladder washings from 59 patients with cancer, and 195 symptomatic control patients were collected. Cells were double-labeled with M344 monoclonal antibody and Hoechst. Each case was blinded, and the number of positive cells was scored by two independent observers. High-grade and low-grade transitional cell carcinomas (TCC) were detected with equal efficiency (78%, P < 0.001 versus symptomatic control patients). Urine samples proved higher specificity in detecting cancers. Patients being monitored for recurrence, but without current detectable cancer, were intermediates between control subjects and patients with cancer, suggesting that this marker also responds to dysplasia or field disease. Patients with outlet obstruction did not significantly differ from patients with previous TCC (P = 0.95). When combined with DNA ploidy measurements and cytology, the sensitivity for low-grade and high-grade tumors was 88% and 95%, respectively. The M344 antibody potentially could improve the specificity and sensitivity of detection of low-grade bladder tumors in symptomatic and asymptomatic patients as well as monitoring for recurrence, therapeutic response, and assessment of individual risk.

The Effect of Recombinant Cytokines on Bladder Cancer Cells in Vitro

We have studied the Major Histocompatibility Class II-modulating and antiproliferative actions of recombinant interferon-γ, tumor necrosis factor-α, interleukin-1α, interleukin-2 and granulocyte macrophage-colony stimulating factor on bladder cancer cells in vitro. Indirect immunofluorescent staining employing monoclonal antibody probes in conjunction with flow cytometric analysis and tritiated thymidine incorporation assays were used.Interferon-γ was a strong inducer and enhancer of class II antigens. Tumor necrosis factor-α could not induce the expression of class II antigens on tumor cells, which were initially class II-negative, but enhanced the expression on cells which already demonstrated low levels of the antigen. Interleukin-1, interleukin-2 and granulocyte macrophage-colony stimulating factor did not affect the major histocompatibility class II expression.Interferon-γ and tumor necrosis factor-α both demonstrated powerful anti-proliferative effects on the bladder cancer cells, particularly those derived from low grade tumors (G1 and G2). Interleukin-1α had no effect on tumor cell proliferation. In contrast interleukin-2 and granulocyte macrophage-colony stimulating factor had significant stimulatory effects on the proliferation of the low grade tumor cells (G1).

Molecular and Cellular Biological Approaches and Techniques in the Detection of Bladder Cancer and Enhanced Risk for Bladder Cancer in High-Risk Groups

Recent advances in molecular cell biology, immunology, and toxicology enhance detection of actual and incipient disease and the definition of risk. Tumor-associated antigens may greatly improve detection of low-grade tumor cells. Several new strategies depend upon detecting molecular lesions of the genes controlling cell growth (oncogenes) at either the gene or protein levels. Highly sensitive techniques detecting DNA adducts are refining the ability to detect specific DNA damage shortly after exposure. The recognition of controls that actively stop proliferation (tumor suppressor genes), regulate differentiation or of relationships between metabolism and carcinogenesis may lead to new ways to identify persons genetically at increased risk from carcinogenic exposure. Current technologies, those on the near horizon, and long-term research needs are analyzed critically in terms of screening objectives.

Limitations of flow cytometric DNA analysis for the diagnosis of bladder cancer.

Flow cytometric analysis was done on exfoliated urothelial cells from 11 control patients and 31 patients with transitional cell carcinoma: 9 grade I, 11 grade II, and 11 grade III. The determination of aneuploidy by DNA analysis did not provide identification of low-grade tumor cells. Other flow cytometric parameters of cellular change such as cell size, nuclear size, nuclear/cytoplasmic ratio, or cell refractivity may provide better identification of low-grade lesions.