Histochemical probes for the detection of hypoxic tumour cells.

Hypoxia is thought to be a major cause of failure in cancer treatment. In this paper, we report methods transposable to clinical practice, for identifying hypoxic tumour cells. They consist of histochemical tests for revealing lactate dehydrogenase activity, endogenous lactate and accumulation of neutral fat. An ascites tumour (Yoshida hepatoma) and a solid tumour (Ehrlich carcinoma) were used as the experimental models. A gel film technique was used for visualizing lactate dehydrogenase and "nothing dehydrogenase" (or endogenous lactate). The fluorescent dyes Nile Red and Acridine Orange were used to demonstrate lipid accumulation and to visualize the tumour morphology, respectively. Tumour cells at the edge of areas of necrosis and at a distance of about 130 microns from a blood vessel were presumed to be hypoxic and showed the following features: 1) a dark blue granular pattern of lactate dehydrogenase (LDH) activity, ascribed to intense activity of the LDH5 and/or LDHk isoenzymes bound to membranous structures; 2) an intense granular positivity of "Nothing Dehydrogenase" due to high concentrations of endogenous lactate; 3) neutral lipid droplets emitting an intense yellow fluorescence in Nile Red-stained preparations; 4) a yellow cytoplasmic fluorescence in Acridine Orange-stained sections, attributable to a low cellular RNA content. Electron microscopy revealed moderately osmiophilic lipid globules in close association with damaged mitochondria. Better oxygenated cells showed: (a) a reddish-blue diffuse pattern of LDH, ascribed to moderately active soluble LDH isoenzymes containing H subunits; (b) almost no "Nothing Dehydrogenase" positivity; (c) no cytoplasmic lipid droplets; and (d) an intense orange-red fluorescence in the cytoplasm of Acridine Orange-stained specimens, due to high concentrations of cellular RNA. Nile Red fluorescence showed that the lipids of the solid tumour membranes were more hydrophobic than in the normal surrounding tissue. This suggests that there are abnormal domains of neutral lipids in the tumour cell membranes. In solid tumours, cells with the characteristics attributable to hypoxia were usually observed on the edge of necrosis of cuff-like formations. In very advanced growth stages, however, they were also seen surrounding (and occasionally clogging) blood vessels, or in tentacular formations coming from a necrosis border and polarized towards the vessels. Lipid-loaded cells were also seen in blood vessels distant from the tumour. These observations point towards a chemotactic process of hypoxic cells towards better environments.

Neoplastic cell spreading in rodent transplantable tumor models. I. Ehrlich tumor.

Ehrlich ascites tumor cells were ectopically transplanted in femoral muscles of tumor-free Swiss and BALB/c mice with the same modality used for i.p. serial transplantations of the ascitic form. A solid tumor developed (100% takes as i.p. grafts) locally invading surrounding tissues and leading to death within 30-40 days (12-14 days in ascitic form). These animals were killed when showing signs of debilitation by tumor growth (1 mo.). The recipients' own thoracic and abdominal organs (lung, liver, spleen, and kidney plus peritoneal fluid) as well as the solid tumor were removed to obtain imprints and smears fixed and stained for cytology (May Grünwald Giemsa). Tumor-free mice were used as a control and i.p. transplanted mice were sacrificed on day 8. Disseminated tumor cells were seen in recipient organ imprints and peritoneal fluid smears scattered among local normal cells. Host defense cells with prevalence of neutrophils were observed infiltrating the solid tumor or adjacent to disseminated tumor cells. According to previous findings, organ imprints of i.p. transplanted mice showed disseminated tumor cells and host defense cells. Surprisingly, in liver imprints of ectopically transplanted BALB/c mice, numerous megakaryocytes were detected. This tumor and host organ imprint assay offers the possibility to monitor in vivo the phenomenon of metastatic tumor spread.

Neoplastic cell spreading in rodent transplantable tumor models. II. Santamaria tumor (syngeneic keratinizing squamous cell carcinoma).

A syngeneic transplantable tumor was obtained in our laboratory by inducing a skin squamous cell carcinoma in BALB/c mice treated with benzo(a) pyrene and UVA. Single tumor cell suspensions obtained by finely disrupted tumor masses were either i.p. or intramuscularly injected and developed (100% takes) invasive tumors maintaining in subsequent analogue serial transplantations identical histopathological aspects. May Grünwald Giemsa stained organ imprints of tumor bearing mice showed disseminated tumor cells as well as a number of infiltrating host defense cells (principally neutrophils) despite the mice were in a terminal status. May Grünwald Giemsa stained cryostat sections showed numerous mast cells lining the invasive tumor front and allowed to detect in the liver tumor cells migrated from primary tumor (localized in femoral muscle) adhering to endothelial cells may be to perform extravasation.

Morphological characterization of lymphoid cells conjugates with scattered tumor target cells in organ imprints of Yoshida ascites bearing rats.

The authors analysed morphologically the ability of host effector cells to bind disseminated tumor target cells (pre-lysis) in vivo. Scattered tumor cells are morphologically visible stained with May Grünwald-Giemsa in the lung, liver, kidney, and spleen imprints of Yoshida ascites tumor bearing rats. Lymphoid cells can be seen binding disseminated tumor target cells in organs but not in the peritoneal cavity. This model may provide a useful technique for morphological studies on the in vivo conjugation capacity of host effector cells against tumor target cells.

Intraperitoneal transplantation of Yoshida ascites hepatoma cells after fine needle aspiration from tumor bearing rats organs.

The authors tested the ability of scattered tumor cells to re-form a tumor in vivo. Disseminated tumor cells are morphologically visible stained with May-Grünwald Giemsa in the lung, liver, kidney, and spleen of Yoshida ascites tumor bearing rats. Free tumor cells can easily be fine needle aspirated from those organs and injected in syngeneic Wistar rats. All the host rats show ascites tumor take after intraperitoneal transplantation of each aspirated sample. This biological model might be useful to study in vivo a wide range of properties of neoplastic and non-neoplastic host cells.

[Preliminary findings on tumor cells in various organs of rats after transplantation of Yoshida hepatoma ascites cells]. / Sul reperto precoce di cellule tumorali in organi diversi di ratti dopo trapianto di cellule di epatoma ascite di Yoshida.

In previous papers the techniques for the study of the migration pattern of tumor cells through the host tissues were described. In the present communication studies utilizing Yoshida ascitic tumor cells are referred. Neoplastic cells morphologically identified were counted in several organs at various times of the development of the tumor transplantation. This method appears to provide interesting indications about the dynamics of the neoplastic spread through the tissues.

Histoautoradiographic investigation of the invasive dissemination of Ehrlich ascites tumor cells through the organs of the mouse.

In this paper the attempt has been pursued to quantify by a well planned experimental technique the ability of the neoplastic cells to spread around in vivo through various organs of laboratory animals. By making use of the histoautoradiographic techniques the incorporation of 3H thymidine either by the normal and by the neoplastic cells has been investigated at many different anatomical sites in Ehrlich ascites tumor-bearing mice. The method has been shown to be well suited also for the quantitative characterization of the spreading dynamics of the cancer cell population.

[Cytochemical study of cells of primary and disseminated ascite Yoshida tumor cells]. / Studio citochimico di cellule di tumore ascite di Yoshida primitive e disseminate.

The different distribution of cytochemically demonstrable enzymes: lactate dehydrogenase (LDH, 1.1.1.27), succinate dehydrogenase (SDH, 1.3.99.1), dihydrofolate reductase (DHFR, 1.5.1.3), acid phosphatase (AcP, 3.1.3.2) and alkaline phosphatase (ALP, 3.1.3.1), has been documented in Yoshida ascites hepatoma cells in vivo or stored at 80 degrees C. The dehydrogenase activities (LDH, SDH, DHFR) show a strong reaction in all samples. An increased level of these enzyme activities has been observed in the malignant cells spreading through the organs of tumor bearing rats. On the contrary, in the same samples, acid and alkaline phosphatase activities are very low. The strong dehydrogenase activities observed in Yoshida ascite cells stress the rapid turnover of tumor cells. Our results indicate that the histochemical method may be a useful tool to detect the scattered tumor cells. Furthermore, the cytochemical methods allow the characterization of the metabolic pathways employed by the primary and disseminated tumor cells.

Kinetics of the spread of the Yoshida ascites hepatoma cancer cells through various organs of the rat.

In the present paper the attempt is pursued to provide a self-consistent interpretation for the quantitative measurements of the amount of neoplastic cells found at different anatomic sites and at various times after intraperitoneal injection. The model, employing a system of 8 linear differential equations with constant coefficients, can be fitted by means of a special program for a small processing system equipped with an 8-bit microprocessor and 48 KB of central memory.