Deep sea sedimentation

An important problem in the study of microparticles in the marine environment, suspended in the water column or deposited as sediment on the ocean bottom, is the determination of provenance of the microparticles—where did they come from and by what processes were they transported to the sampling location? Two techniques of possible interest to those concerned with tracing the origins and dispersion paths of asbestos particles are described. One utilizes variations in the naturally occurring rubidium–strontium isotope system and is used to characterize a bulk sample, i.e., a large number of particles. The other utilizes scanning electron microscopy to observe variations in surface texture of individual grains which, in the case of quartz particles in the natural environment, can be related to the transport processes to which they have been subjected.

Human breast cancers have proved to be among the most difficult tumours to grow in immunodeficient mice. Two large scale attempts to establish human breast tumour xenograft lines from fresh tumour specimens have been successful for only 6.5% of 433 specimens (Giovanella et al., 1991) and 9% of 93 specimens (Fogh et al., 1982), with no evidence of metastatic growth reported.
This report describes a human breast tumour xenograft line originating directly from the patient specimen which consistently metastasises to the lungs of athymic nude mouse hosts (Hurst et al., 1991).

Materials and methods
Human mammary tumour xenograft line The mammary tumour line (GI-101) was derived from a local first recurrence of an infiltrating ductal adenocarcinoma (Stage Illa, T3N2MX) in a 57 year old female who had not received any therapy other than surgery. The tumour was implanted by trochar in 3-5mm3 pieces subcutaneously to the subaxial area of 12 week old (NCr) athymic nude female mice. The GI-101 tumour line has been maintained for the past 8 years by serial trochar transplant from 1,500-2,000 mm3 tumours.
Recipient animals are kept in a pathogen free environment and are negative for pathogenic murine bacteria and viruses. All husbandry and experimental procedures are performed under a Class B laminar flow hood. Tumour histology and lung metastases are monitored for each transplant generation.
Animals are maintained according to ILAR guidelines. All procedures involving animals are performed according to protocols approved by the Animal Care and Use Committee at the GICR in compliance with PHS guidelines on Animal Welfare Assurance.
Measurements ofprimary tumour Xenografted subcutaneous tumours were measured once weekly by vernier caliper and the volume calculated as a hemi-elipsoid using the formula 0.5 (length x width x thickness). Immunohistochemistry All immunohistochemistry (except for (ER) oestrogen receptors and (PR) progesterone receptors) was performed on neutral, buffered formalin fixed, paraffin-embedded tissues. Immunostaining with Mabs was performed with the Vectastain Elite ABC kit with DAB as the chromogen. Endogenous murine IgG did not cause significant background staining when using formalin-fixed, paraffin embedded sections. ER and PR receptors were detected with Abbott and CAS., Inc. kits, respectively. Flow cytometric DNA profiles Single parameter flow cytometric analysis was performed as previously described (Schiano et al., 1991). Briefly, mechanically-disrupted or enzyme-dissociated cells were stripped of cytoplasm with a detergent and DNA stained with propidium iodide. An EPICS 541 Flow Cytometer (Coulter Corp.) with an argon laser was used to measure DNA. Twenty thousand cells each, from GI-101 human breast tumour, mouse spleen and human peripheral blood lymphocytes were analysed.

Results
Average growth rates of different transplant generations of GI-101 are depicted in Figure 1. Individual tumour growth rates within a single transplant generation group (5-10 animals) were variable although the size of tumour implants was approximately the same. Whether this variability reflected differences in physiological status of the host and/or the tumour implant remains to be determined.  The GI-l01 xenograft line has remained consistent as a a poorly differentiated mammary carcinoma with occasional acinar and ductule formation (Figure 2a). The stromal component is significant and in frozen sections stains intensely for murine immunoglobulins. Although the tumours rarely ulcerate, large tumours usually contain a necrotic core. Lung metastases have been observed from the first transplant generation and consist of multiple foci of undifferentiated cells (Figure 2b). Of the specimens examined, metastatic lung foci were rarely apparent until the subcutaneous implant had grown to a volume of 500 mm3. Metastatic lung foci may replace large segments of normal tissue by the time the implant has grown to 2000 mm3. The size and number of lung metastases are approximately proportional to tumour size (data not shown). Tumour take rate is generally 100%. Both breast tumour implant and lung metastases showed strong affinity for Mabs to several breast tissue differentiation antigens which are listed in Table I. Both tissues also stained lightly to moderately positive for human b cytokeratins (AEI/AE3), recombinant cathepsin-D, and the tumour-associated antigens targeted by the Mabs 1a5-6A, B72.3 and p53. Primary tumour and metastases were negative for nuclear proliferating antigen, breast cystic disease fluid, carcinoembryonic antigen, 17-lA, and CC49 (a chimaeric subclone of B72.3). Tumour and metastases were also negative for both internal and external domains of c-erbB-2 oncoprotein and for ER and PR.
Control diploid human peripheral blood lymphocytes generated DNA peaks at channels 80 (GI) and 157 (G2), while control diploid mouse spleen cells showed DNA peaks at channels 74 (GI) and 138 (G2) (Figure 3). A comparison of histograms from GI-101 breast tumour and control histograms demonstrates the presence of both murine and human components. Breast tumour cells were all hyperploid, displaying GI phase DNA peaks at channels 98-100. The coeffih cient of variation for channels was no more than ±3 chans tnels.   study. Anaplastic lung metastases showed the same affinity for normal breast tissue markers as the more differentiated primary tumour. The human genotype of the tumour has been ascertained both by flow cytometric DNA profiles and positive immunohistostaining with Mabs against human breast tissue-associated antigens, none of which cross reacted with control normal murine tissues. GI-101 is negative for the c-erbB-2 oncogene, but positive for p53. Mutation of the p53 gene (important in the initial events controlling cell division) is the most common genetic change observed in breast and other cancers, and is associated with a more aggressive phenotype (Harris, 1992).
DNA flow cytometry profiles of breast tumour showed that it contains both murine and human components, the tumour cells being mainly in the GI phase with a small proportion in G2 and S phases. Although the diploid murine cell population in this tumour has not been identified, Mab against murine IgG in frozen sections was confined to the tumour stroma, being completely absent in epithelium. Further experiments designed to explore the above observations and to evaluate the role of the murine stromal component in xenograft growth and metastases are underway.