The development of cancer is characterized by a long latency between the initial exposure to a carcinogenic substance and the final appearance of a macroscopic tumor. Histochemistry has provided the means to identify specific cells involved in this process and many of the changes that take place during the transformation of normal cells to malignant ones. Putative premalignant lesions have been studied extensively in liver carcinogenesis. Histological sections of grossly normal appearing liver a few weeks after treating animals with carcinogen contain discrete areas or “islands” of aberrant enzyme activity that can be detected with enzyme histochemistry and are commonly called “enzyme-altered foci.” These foci display altered activity of one or more enzymes; one of the most common is increased γ-glutamyl transpeptidase (GGT) activity. Since the activities of most enzymes are destroyed by the heat and solvents used for paraffin-embedding, most of this work was done with frozen sections that are often 10 μm in thickness

Three cell types not previously described were revealed by the application of several microscopic procedures in combination with cytochemistry and irnmunocytochemistry in the livers of rats treated according to the Solt et al carcinogenesis protocol. This protocol consists of the administration of an initiating carcinogen (diethynitrosamine), a mitoinhibitory carcinogen, (acethylaminofluorene) and a growth stimulus (partial hepatectomy) (1). Two of the cell types were found in intrahepatic bile ductules and one within the connective tissue stroma surrounding the ductules. These findings have implications for understanding the cell lineage pathways that operate in an experimental rodent hepatocarcinogenesis system in which hepatocyte regeneration is inhibited and in which preneoplastic nodules and hepatomas develop. Our previous studies have determined some of the enzymatic, antigenic and structural properties of these cell types and their interrelations to each other, to parenchymal hepatocytes and to preneoplastic nodules (2).

Proliferation of bile ductule cells occurs early (24-48 hrs) after the partial hepatectomy step of the protocol with extensive branching of the ductules into the hepatic parenchyma.

Although ovarian carcinomas are the most lethal gynecologic tumors, their origins remain unclear. Do they develop from malignant transformation of benign neoplasms through a multistep process of tumor progression, or do they arise de novo? The histologically benign and/or low malignant potential (LMP) components in heterogeneous ovarian carcinomas have been considered as evidence supporting the theory of tumor progression. These components are interpretted as the remnants of pre-existing neoplasms that underwent malignant transformation. In two other possible interpretations, however, such components may be clones which developed independently (de novo hypothesis) or they may represent malignant epithelium which underwent focal maturation (maturation hypothesis).

To evaluate genetic relationships of the histological components in heterogeneous ovarian carcinomas, 10 such neoplasms and 5 normal ovary controls were examined using fluorescence in situhybridization (FISH) on intact paraffin sections (6 μm).The retention of tissue architecture allowed direct correlation of detectable genetic aberrations with histology, and comparison of malignant components with adjacent histologically benign or LMP components.

The presence of estrogen and progesterone receptors on human breast carcinomas correlates with both response to hormonal therapy and survival. Quantitation of receptor activity is typically performed by radioreceptor assay on fresh frozen tumor tissue obtained at the time of surgical resection. Estrogen and progesterone receptor reactivity can also be detected immunohistochemically on frozen sections of breast cancer and in appropriately fixed paraffin embedded tissues. Several studies have shown that semi-quantitative visual determination and quantitative image analysis assessment of estrogen and progesterone receptor immunoreactivity on fixed tissue sections is highly correlated with biochemical measurements of receptor in frozen samples. We recently developed a procedure for the simultaneous localization and quantitation of antigens in fixed tissue samples that does not involve antigen extraction, radioactive materials, or image analysis. In this procedure, which we term a “Midwestern assay”, fixed tissue sections are used with antigen-specific enzyme-linked antibodies to generate soluble reaction products which are spectrophotometrically quantitatable, as in an ELISA, and deposited reaction products which are microscopically localizable, as in enzyme-based immunohistochemistry.

We have described previously the presence of IL2R on human carcinoma (CA) cell lines (squamous cell carcinoma of the head and neck, gastric carcinoma, renal cell carcinoma) and tumors in situ (1,2,). These carcinomas expressed IL2Rβ and γ (intermediate affinity IL2R) and I-labeled IL2 was found to bind to these receptors. Scatchard analyses showed that 13,500 IL2Rβ/cell and about 300 IL2Rα were present on the surface of PCI-1, a SCCHN cell line. The mRNA and proteins for IL2Rβ and γ were consistently detectable in tumor cell lines and fresh tumor tissues. Binding of recombinant IL2 to IL2R resulted in a partial but significant inhibition of tumor cell growth, decreased expression of class I MHC antigens and ICAM-1 on tumor cells and their increased sensitivity to growth-modulating cytokines IFN-γ, TNF-α or TGFβ. These effects were IL2-dose dependent, and were optimal at doses >10nM. Anti-IL2Rβ Abs reversed IL2-induced growth inhibition, an indication that exogenous IL2 delivered a negative growth signal to CAs via the β chain of IL2R.

Poly-[(trans-1,2-diaminocyclohexane) platinumj-carboxyamylose (“poly-plat”), 5-sulfosalicylato-trans -(1,2-diaminocyclohexane) platinum (SSP), and 4-hydroxy-∝-sulfonylphenylacetato (trans 1,2-diaminocyclohexane) platinum (II) (SAP) are second generation analogs of cisplatin (CDDP) with higher efficacy and potency than cisplatin. This is particularly true of “poly-plat” which contains 1/5 the platinum of CDDP. In order to understand the mechanism of action of these compounds, isolated murine peritoneal macrophages in culture medium were treated with “poly-plat”, SSP, or SAP (5 μg/ml) for 2 h. Drug containing medium was then replaced with fresh medium and the cells were allowed to incubate at 37° C (5% CO2) for 24 h. Supernatants were collected at 0.5, 1, 2, and 24 h post-treatment for immunocytochemical analysis. Confocal microscopy studies demonstrated an increase in the number of lysosomes in the treated macrophages, but only “poly-plat” and SSP treated macrophages were stimulated to form cytoplasmic extensions at 2 h and 24 h.

Immunohistochemistry (IHC) has provided major insights about the classification of brain tumors by identifying cellular markers of phenotype, and about tumor growth potential with nuclear markers of proliferation. Newer in situ hybridization shows promise in tumor classification and prognostication.

Fig. 1 shows the sensitive and reliable avidin-biotin conjugate (ABC) method of localizing glial fibrillary acidic protein (GFAP). GFAP is the most specific marker of gliomas, tumors of brain cells, available today. The ABC method can be used to find any antigen for which a primary antibody is available. A molecular bridge then links this primary antibody bound to tissue with a label that can be seen, a substrate of the horseradish peroxidase (HRP) enzyme like diaminobenzidine (DAB) that produces a brown, insoluble reaction product at the site of the antigen. IHC for GFAP has revealed that brain tumors previously thought to be sarcomas are actually malignant gliomas (Fig. 2).

The human immunodeficiency virus (HIV-1) is dependent upon the synthesis of TAT for efficient transcriptional activation and replication. Following translocation to the nucleus TAT interacts with a cis-acting TAT-responsive element located between nucleotides +1 and +42 within the HIV-1 long terminal repeat. This region contains an RNA stem loop structure TAR which interacts with TAT and cellular proteins to increase transcription 1000 fold We have transfected human T-lymphocytes with an expression vector containing the HIV-1 LTR driving the synthesis of TAT-72. A stable cell line was generated by selection with G418. Cells were grown on round glass coverslips coated with a solution of fibronectin overnight (at 37 °C). In addition to light microscopy (LM) observations, cells on coverslips were fixed with a 3.5% glutaraldehyde buffered solution (0.1 M Na cacodylate) for 5 min at room temperature and gently centrifliged in a microfuge (400 rpm, 3min) to obtain a cellular pellet.

Large cell (T - cell) lymphoma is a malignant neoplasm derived from activated lymphoid cells, involving lymph nodes, spleen, subcutaneous lesions. Ultrastructural examination facilitates the determination of this particular type of lymphoma, specifically by the presence of lytic granules in the cytoplasm containing proteins involved in the target cell destruction. One of these, expressed specifically is TIA-1, a cytotoxic granule-associated protein with RNA binding domains initially identified using a monoclonal antibody reactive with a 15KD protein present in the granules of cytotoxic lymphocytes.

Tissue from seven cases of large cell lymphoma from the archives of our department were fixed for electron microscopy studies with a mixture of4% paraformaldehyde and 1% glutaraldehyde in 0.1M sodium cacodylate buffer and embedded either in Poly/Bed 812 with osmium tetroxide postfixation, or in LR White without osmication. For immunogold labelling, ultrathin sections placed on Nickel grids were incubated with a murine monoclonal TIA-1 antibody (1:50) (Coulter Co) overnight at 4°C.

Malignant tumors are known to have an heterogeneous cell population. Metastases are supposed to be formed by subpopulations with high metastatic capability. These metastatic cells constitute the so called invasive phenotype. In that order of ideas, metastases could be pathologically different from their parent tumors if the invasive phenotype had distinct morphological features. The similarities or differences between primary tumors and their metastases have not been adequately studied at the ultrastructural level. In this work we report an electron microscopic study of liver leiomyosarcoma metastases which shows alterations not described in primary tumors of that kind.

Biopsies of liver metastases from a colon leiomyosarcoma were surgically obtained. Samples were processed with routine techniques for transmission electron microscopy and observed in a Hitachi H-500 electron microscope.

Some alterations usually observed in primary leiomyosarcomas were seen. They were presence of myofilaments with focal densities, nuclear changes, swollen mitochondria, and abundance of rough endoplasmic reticulum (Fig. 1).

C-ret proto-oncogene encodes a protein in the tyrosine kinase (TK) family of transmembrane receptors for growth factors. C-ret expression has been identified in normal tissues and tumors of neural crest origin such as inherited MEN (multiple endocrine neoplasia) syndromes, particularly medullary thyroid cancers where there are confirmed germ-line mutations. Gene rearrangement has been observed in papillary thyroid carcinomas (PTC) and has been shown to have cytoplasmic localization of the altered gene product. C-ret proto-oncogene product is also necessary for the normal development of the peripheral nervous system, enteric nervous system and the kidney. A recent profile of the TK receptors in prostate cancer (PCA) identified c-ret protein in prostate cancer tissue and xenograft derived from human PCA. This study is the first to report the immunohistochemical (IH) evaluation of c-ret in human prostatic tissue.

Thirty radical prostatectomy specimens for PCA were formalin-fixed, sectioned at 5mm and paraffin-embedded.

Vitamins C (VC) and K3 (VK3) and a VC: VK3 combination with a VC: VK3 ratio of 100:1 were assayed for their antitumor activity against a DU 145 human prostate cancer cell line. Co-administration of the vitamins enhanced the antitumor activity 20-25 fold even with a lh exposure time. While exogenous catalase destroyed the antitumor activity, hydrogen peroxide-induced lipid peroxidation was negligible. The results of additional assays revealed: a transient increase in ATP production, a decrease in DNA synthesis, and increase in protein synthesis and a decrease in thiol levels. Subsequently the DU145 cells were treated with each vitamin alone or with the vitamin combination at their 50% cytotoxic doses and then examined using transmission electron microscopy. Treatment of DU145 cells with the vitamin combination exacerbated the cytotoxic effects induced by individual vitamin treatment.

Untreated DU145 cells show numerous mitochondria whose pale matrices are in sharp contrast to the cytoplasmic background (Fig. 1).

Laser-induced fluorescence (LIF) spectroscopy of tissues has been proposed as an adjunct to endoscopy for in vivo gastrointestinal (GI) diagnosis of premalignant lesions. In previous studies, under ultraviolet excitation (350-370 nm), sources of LIF signal differences in normal, and premalignant colonie tissues have been identified including 1) changes in gross tissue morphology, 2) increased hemoglobin absorption in adenomatous tissues, and 3) increased red fluorescence in dysplastic cells. However, many questions remain regarding the specific origins and biochemical correlates of GI tissue autofluorescence.

We report on a study of quantitative ultraviolet LIF spectral microscopy in human colonic tissues using a laser scanning confocal microscope with argon laser excitation 351-364 nm. Frozen sections (6μm) of normal colon (n=10) and tubular adenoma (n=8) were prepared from fresh surgical resection specimens using a previously published protocol. To identify histological components accurately, LSCAM slides were alcohol fixed and H&E histologically stained following confocal imaging.

Identification of altered gene expression in colonie preneoplastic lesions may play a key role in early detection and prevention of colon cancer. Considering carcinogen treated rat colonie mucosa harbors a mixed population of both normal and aberrant crypts, it is of interest to independently examine the genetic profile of these two populations. The present study was designed to use a novel method whereby RT-PCR was performed on alcohol fixed ACF which were micro dissected from normal surrounding crypts.

Male F344 rats were injected with azoxymethane, a colon specific carcinogen, at a dose of 15 mg/kg body weight once weekly for 3 weeks. After 28 weeks colons were excised, flushed with RNAse free ice cold phosphate-buffered saline, fixed flat on filter paper, fixed in 70% ethanol and immediately frozen at -80°C After a minimum 24 hours colons were placed on the lid of a sterile, clear petri dish filled with ice.

There have been considerable interests in the relationship between tumor cell proliferation and angiogenesis. In human breast carcinomas, it has been reported that angiogenesis is an independent prognostic factor and that proliferating factors are closely related with lymph node status. Therefore, the demonstration of proliferating and angiogenetic factors as well as the tumor blood vessels in human breast carcinoma simultaneously is important for clinical evaluation of breast cancer patients.

CLSM has been an exciting new apparatus because of it's more powerful resolution than conventional light microscopy and the high-performance on 3D optical sectioning. Images taken with CLSM can be digitized and made variable for image analysis manipulation and computer-assisted 3D reconstructions.

In the present study, we employed immunohistochemical comparative 3D visualization of proliferating and angiogenstic factors with microvessels in human breast carcinomas by CLSM.

The tissues from the surgically resected breast carcinoma were fixed in cold 4 % paraformaldehyde(PFA) and routinely processed in paraffin wax.

To facilitate detection of tumor cells at the highest resolution in an athymic nude mouse model system, Balb/c 3T3 cells transformed with the Harvey ras oncogene were transfected with the histochemical marker gene, bacterial lacZ (LZEJ cells). Alternatively, 3T3 cells transformed with the human sis oncogene were transfected with human placental alkaline phosphatase marker gene (APSI cells). Within minutes of subcutaneous injection, these tumor cells could be detected histochemically and the fate of cells followed with time. APSI or LZEJ cells gave very different single-cell morphologies at this site but yielded similar aggregation patterns of cells. Clearance of some cells could readily be detected by diffusion of histochemical product as “curly-haired” organizations of cells condensed into ovoid collections. Expansion of the population occurred with division of many cells in the population, not just one or a few cells. Cell number was quantitated by the development of ultrasensitive luminometry assays for the two histochemical marker enzymes; different kinetics were observed for establishment of LZEJ or APSI cells.

Metastasis is a highly selective nonrandom process favoring the survival of minor subpopulations of metatastatic cells that preexist within the primary tumor. The cellular and molecular mechanisms that regulate the metastasis of tumor cells to specific organs are diverse and both tumor and organ-specific (1). Many examples exist in which malignant tumors metastasize to specific organs. As Paget proposed in 1889 (2), and as our recent biological and molecular evidence demonstrates, the organ microenvironment influences the invasion, survival, growth, and apoptosis of particular tumor cells. This hypothesis explains metastatic colonization patterns that cannot be due to solely mechanical lodgement/anatomical considerations (1). Successful metastasis therefore involves the interaction of tumor cells with a compatible milieu provided by a particular organ environment.

Recent experimental evidence suggests that paracrine stimulation of tumor cells by organ-derived growth factors and cytokines is one mechanism which determines the target organ preference of disseminated cancer cells.

The formation of vascular stroma (angiogenesis) is essential for tumor growth and may also influence invasion and metastasis. Quantitation of angiogenesis may serve as a prognostic marker in certain malignancies and novel therapies targeted at inhibiting angiogenesis may prove to play an important role in control of tumor growth.

In situ hybridization (ISH) studies have helped to define some of the molecular and cellular events responsible for the formation of vascular stroma in malignancy.

Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), is a multifunctional cytokine associated with tumor angiogenesis. Vascular endothelial cells express at least two tyrosine kinase receptors for VPF, flt-1 and KDR. VPF acts as an angiogenic factor in several ways. It is an endothelial cell growth factor, alters endothelial cell gene expression, induces endothelial cell migration, and it is also a potent inducer of microvascular hyperpermeability (50,000 times as strong as histamine).

Non-invasive imaging tools using biosensors and biomarkers for defining the function of living cells and tissues are among rapidly emerging technologies being developed to monitor cellular toxicity mechanisms. Commercially available fluorescence probes and naturally occurring fluorescent molecules can be used to quantitatively monitor a number of functional endpoints in cultured cells. Innovative approaches to exploit the sensitivity, spectroscopy and temporal/spatial resolution properties of fluorescent probes may provide sensitive approaches for analysis of the molecular mechanisms of toxicity in cells exposed to a variety of toxicants. Kinetic analysis of multiple cellular parameters such as intracellular glutathione (GSH) and Ca2+ content, reactive oxygen species (ROS) generation, mitochondrial and plasma membrane potentials, intracellular pH, and gap junction-mediated intercellular communication (GJIC) with cellular component-specific fluorescent probes permits rapid identification of changes in these parameters and the chronology of injury in cells caused by acute toxicant exposure. Cellular signal transduction mechanisms are also likely to be vulnerable to toxic insults.

Plasmodium falciparum, the most malignant human malaria, is responsible for 2-3 million deaths annually. These infections often involve blockage of the cerebral microvasculature by P. falciparum-infected erythrocytes (Fig. 1). This aspect is considered the major factor in the pathogenesis of cerebral malaria.

Upon invasion of the erythrocyte, P. falciparum immediately begins to remodel the infected erythrocyte. The adherence points of infected erythrocytes, termed knobs (Fig. 2 and 3), contain antigenically diverse 200-350kDa surface proteins (PfEMPl; Fig. 4). The PfEMPl variant surface proteins are encoded by a large and extremely diverse family of genes (var), and switches in the expression of var genes account for rapid changes in the antigenic and adhesive properties of P. falciparum-inkcted erythrocytes (2.4% per generation). Switches in the PfEMPl expression may not only affect the phenotype of the parasite strain but may also change its sequestration to endothelial cells. Genetic reorganization in this protein can lead to binding any of the following endothelial cell receptors; ICAM-1, CD36, thrombospondin, chondroitin sulfate (Fig. 5),2 ELAM-1, or VCAM-1.