Mast cells and eosinophils in invasive breast carcinoma

Background  

Inflammatory cells in the tumour stroma has gained increasing interest recently. Thus, we aimed to study the frequency and prognostic impact of stromal mast cells and tumour infiltrating eosinophils in invasive breast carcinomas.     

Elastases in human breast carcinoma cell lines

Elastosis, the deposition of large amounts of elastin, is characteristic of the desmoplastic reaction to human breast carcinoma. Dissolution of the elastin often occurs following treatment regimens that involve steroid hormones or their antagonists. Elastinolytic activities must be invoked to account for the loss of this elastin-cotaining stroma. We have utilized a tissue culture model to explore the molecular aspects of this phenomenon. The elastases of several human fibroblast and breast carcinoma cell lines were examined. The tumor cells had 10- to 30-fold higher elastase activity than did the fibroblasts. Three separate elastinolytic activities were observed in the tumor cell lines, and partial purification was achieved. The effect of steroid hormones on these elastases was examined. No stimulation of activity was found with any of the hormones, in any combination. However, there was marked inhibition of elastase with estradiol, progesterone, and dexamethasone of the ZR75-1 cell line. This is the estrogen receptor positive line that is estrogen responsive. The corticosteroids also inhibited the elastases of the estrogen receptor positive, non-responder cell line ZR75-30. No effect was seen on the elastases of receptor negative cells ZR75-31A with any of the steroid hormones. Stimulation of elastinolytic activities in these tumor cells must occur by some as yet unidentified pathway.     

Prevention of tumour cell dissemination in diagnostic needle procedures

Background:

A side effect of diagnostic needle biopsies is the possibility to disseminate tumour cells into the needle track, which may cause concern in certain malignant tumour types.

Methods:

In order to prevent tumour cell dissemination we developed a technology that uses radiofrequency (RF) pulses to sterilise the needle track and denaturate tumour cells. To determine feasibility, we applied this technology to fine needle aspiration biopsy (FNAB) and used breast cancer as a model tumour. Routine FNAB was performed in 88 patients with adenocarcinoma and blood droplets passing the skin orifice were cytomorphologically analysed for the presence of tumour cells.

Results:

The analysis showed the presence of tumour cells in 65/88 cases (74%). When using an experimental anti-seeding device in a subset of patients viable tumour cells were found in 0/31 cases (P<0.001). In all 31 patients blood passing the skin orifice was sparse. No degrading effect on the cytological sample inside the needle was detected and pain caused by the RF pulses was comparable to that of the biopsy procedure itself.

Conclusion:

The herein presented method has the potential to prevent the dissemination of viable tumour cells in the needle track and minimize bleeding without additional pain or degradation of the aspirate.     

Collagenases in human breast carcinoma cell lines

The intense stromal response to some human tumors is termed the desmoplastic reaction. It is found with most human breast carcinomas. Dissolution of this response, containing predominantly fibrous proteins such as collagen and elastin, can occur with treatment. We have undertaken a study of the collagenases of the breast tumor desmoplastic reaction using a tissue culture model composed of human breast tumor cell lines and various human fibroblasts. The breast tumor cells had the higher collagenase activity, particularly the ZR75-31A cell line. Activity was 10-fold higher than that of the stromal cells. The enzyme was secreted into the media and required trypsin pretreatment for activity to be manifest. Partial purification was achieved of the major collagenase species. The protein was a metalloprotease and, like other mammalian collagenases, had a relative molecular weight of 60,000. Classical 3/4 and 1/4 cleavage products of the triple helical collagen substrate were demonstrated, typical of most mammalian collagenases. Only types I and III collagens were suitable substrates for this enzyme, with no apparent preference between the two. The breast tumor collagenases were not responsive to hormones; however, stimulation of activity was apparent in the absence of proteolytic pretreatment. This may represent conversion of the procollagenases of the breast tumor cells to the active form by an estrogen-sensitive plasminogen activator secreted by the same tumor cells.     

Impact of tumour size on axillary involvement and distant dissemination in breast cancer

Background:

Tumour size and nodal involvement are the two main prognostic factors in breast cancer (BC). Their impact on the natural history of BC is not fully captured by analyses that ignore their quantitative nature.

Method:

Data pertaining to 18 159 patients treated with primary surgery: 3661 at the Institut Gustave-Roussy (IGR, France) between 1954 and 1983, and 14 498 in the breast cancer registry in the Stockholm–Gotland Health Care region (SG, Sweden) between 1976 and 1999, were collected. The risks of distant metastases (DMs) and of nodal involvement were analysed according to tumour size with parametric models.

Results:

Using SG 1976–1990 as the reference group, relative risks (RRs) for DM were equal to 1.42 (95% CI: 1.29–1.56; P<10⁻¹⁰) in IGR and 0.61 (95% CI: 0.55–0.67; P<10⁻¹⁰) in SG 1991–1999. Differences in tumour size explained the increased risk in IGR (RR adjusted for tumour size 1.09; 95% CI: 0.99–1.20; P=0.07), but not the decreased risk in SG 1991–1999 (adjusted RR: 0.63; 95% CI: 0.57–0.69; P<10⁻¹⁰). The relationship between tumour size and DM risk changed significantly during the 1990s.

Conclusion:

Early diagnosis is sufficient to explain differences in the prognosis before 1990. After 1990, the use of adjuvant systemic therapies is the main reason for the reduction in DM.     

Mast cell modulation of tumour cell proliferation in rat mammary adenocarcinoma 13762NF

Mast cells were shown to accumulate around the periphery of the invasive and metastatic rat mammary adenocarcinoma (MTLn3), and histological evidence of mast cell degranulation was observed during the later stages of this model. To assess the physiological role of mast cells in vivo we have used the mast cell-stabilising compound FPL 55618 applied i.p. daily at 1 mg kg-1 for 23 days. Using groups of 12 rats we have found that this compound inhibited tumour growth at the primary site by as much as 70% in most of the treated animals compared with the control group which received equivalent volumes of saline. When the drug treatment was stopped after 23 days, tumour growth of the test group accelerated over the next 7 days and reached a similar tumour size to that of control animals. Histological studies of the tumour and contiguous host tissue at day 24 of the experiment revealed numerous extra-tumoural mast cells often showing signs of degranulation at several sites around the tumour periphery in the control animals. Such observations were not seen in those animals receiving FPL 55618 where, in contrast to controls, numerous intact mast cells were often seen within the tumour mass. Following cessation of the MC-stabilising treatment progressive mast cell activation was evident within 2-4 days, primarily at the tumour periphery. In vitro studies have shown that drug concentrations equivalent to five times the in vivo dose had no effect on the proliferative rate or viability of the MTLn3 cells. Moreover, the proliferative rate of these cells in culture was significantly increased when exposed to soluble mast cell products. Thus our data indicate that a mast cell-stabilising compound has significant benefits in reducing tumour growth in vivo, an observation which supports the concept that mast cell:tumour cell interactions are important for the growth and invasive properties demonstrated by this model of breast carcinoma.     

Increased expression and altered location of annexin IV in renal clear cell carcinoma: a possible role in tumour dissemination

The proteome of renal cell carcinoma and non-neoplastic kidney tissue was analysed from 12 patients by two-dimensional polyacrylamide gel electrophoresis to search for differentially expressed proteins in the tumour. Annexin IV was identified to be up-regulated in tumour cells. These patients and further 11 were characterized by RT-PCR. We found an increased amount of annexin IV mRNA. Immunohistochemical analysis revealed an altered localization of annexin IV in tumour cells. Additionally we demonstrate that over-expressed annexin IV promotes cell migration in a carcinoma model system. From these results above it seems possible that annexin IV plays an important role in the morphological diversification and dissemination of the clear cell renal cell carcinoma.     

Variability of cell size in primary and metastatic human breast carcinoma

Macroscopic measurements and histometrical estimates of cell cytoplasmic (Cx) and nuclear areas (Nx) were made on 24 primary and 37 metastatic tumors from patients with human breast carcinoma. Within the small metastases, the cancer cells were smaller and showed greater variation in size (CVC, CVN) than those in their respective primary tumors. In the larger metastases, the cells were larger and more uniform in size. Studies of primary tumors showed that both Cx and Nx of tumor cells bore a direct relationship to the gross size of the primary tumor. Comparisons of measurements from the 14 primary tumors with metastases and the 10 primary tumors with no metastases revealed no significant differences between the groups. In all tumors, Cx bore a predictable and direct relationship to Nx.     

Granular cell tumour of the breast

Four cases of granular cell tumour of the breast reflecting the lifetime experience of one surgeon are presented. Recent advances in the pathology of this condition suggest that the customary term 'myoblastoma' is inappropriate, and that 'granular cell tumour' is preferable. The clinical and pathological features are reviewed and the differential diagnoses discussed, with emphasis on the distinction from carcinoma which may be mimicked both clinically and on frozen examination. The diagnosis may be made by pre-operative aspiration cytology, and a conservative surgical approach is recommended.     

Granular cell tumour of the breast

Granular cell tumour of the breast (GCTB) is a rare tumour which arises from Schwann cells. It is a largely benign tumour but in extremely rare cases can exhibit malignant characteristics. It posses a particular problem as it's characteristics can mimic breast carcinoma clinically, radiologically and macroscopically. This results in the potential misdiagnosis of breast carcinoma and over treatment of patients. Typically GCTBs are benign, solitary lesions but variations include malignant GCTBs, colocalisation with breast malignancies and multicentricity. These tumours can be investigated using mammography, ultrasound and magnetic resonance imaging. However none of these modalities have yet identified any GCTB specific characteristics. On pathological examination they can be identified using both microscopic and immunohistochemical features. The cells have a distinctive granular eosinophilic cytoplasm associated with typical nuclei and abundant lysosomes. Immunohistochemically they are positive for S100 protein, CD68 and neuron specific endolase (NSE). They are treated with wide local excision and while they may reoccur, are associated with a good prognosis.     

Chemokines induce migrational responses in human breast carcinoma cell lines

Chemokines have been shown to chemoattract and activate different leukocyte populations. Here we report the in vitro effect of macrophage inflammatory protein (MIP)-1α, MIP-1β, regulated on activation, normal T-cells, expressed and secreted (RANTES), monocyte chemotactic protein-1 (MCP-1), interleukin-8 (IL-8), interferon inducible protein-10 (IP-10), neutrophil-activating peptide-2 (NAP-2), growth-related protein (GRO)-α and GRO-γ, on the migration of 3 human breast carcinoma cell lines, MCF-7, T47D and ZR-75-1, using a microchemotaxis chamber to assess migration across fibronectin-coated polycarbonate membranes. MCF-7 cells responded chemotactically to all chemokines tested in a pattern which was dose and time dependent, although responses to the different chemokines were variable. ZR-75-1 responded to MIP-1β and GRO-α, giving maximum migration indices of 3.7 and 5.3, respectively, and exhibited a migratory response to MIP-Iα, IL-8 and MCP-1 although to a lower degree. T47D was unresponsive to the chemokines tested, but both MCF-7 and T47D cells bound radiolabelled ligands with binding constants (Kd) ranging from 0.6 to 2.2 nM and 0.6 to 2.1 nM, respectively. The specificity of the chemotactic response of MCF-7 to MIP-1α and MIP-1β was confirmed using chemokine-specific neutralising antibodies and heat denaturation, and was demonstrated to involve G protein and cyclic AMP signalling pathways. MIP-1β and MIP-1α were shown to induce changes in the organisation of the actin cytoskeleton and the level of F-actin in MCF-7 cells, as determined using flow cytometric analysis and confocal microscopy. Our results show that breast carcinoma cells can respond to chemokines, and suggests a potential role for these molecules in the process of tumour cell migration, invasion and metastasis. Int. J. Cancer 71:257–266, 1997. © 1997 Wiley-Liss, Inc.     

Gender influences tumour growth in a model of human oral squamous cell carcinoma

The pattern of tumour growth was investigated using our unique syngeneic animal model of human oral squamous cell carcinoma of the head and neck (SCCHN). Gender-conditioned tumour fragments of near equal size were transplanted into recipient animals and the pattern of tumour growth was assessed by monitoring tumour size in vivo and/or the weight of tumour al termination of the experiment. Our results confirmed the epithelial nature of tumour cells on fresh tumour tissue sections using morphological and immunocytochemical examination. The mean weights of tumour per animal (n=10) after transplant from female (F) to F recipients, and across and between same gender showed differences if the animals were sexually mature. The results indicated an endocrine sensitive (sex hormone) component of tumour growth. In separate experiments, the recipient animals were immunised with a mechanically dissociated single cell suspension from fresh tumour after irradiation (500 cGys). The test,group (n=5) and control group (n=5, saline injected) animals received tumour fragments by grafting and following a Eur ther three weeks post immunisation, the tumours were removed and weighed. The test and control groups were different with a p-value of 0.001, suggesting an immune participation in the growth of this tumour. The results, taken together, thus suggest the participation of both the endocrine and the immune systems on the pattern of tumour growth in this animal model. The possible clinical relevance of these results is discussed.     

Tumorigenicity and metastasis of human breast carcinoma cell lines in nude mice

There are few reports describing experimental models of the growth and metastasis of human breast carcinomas. This article discusses the tumorigenic and metastatic properties of two estrogen receptor-negative breast carcinomas injected into nude mice. Tumor growth in the mammary fatpad (m.f.p.) and the subcutis was compared in female nude mice. The injection of 10(5) viable cells of two human breast carcinoma cell lines (MDA-MB-231 and MDA-MB-435) gave a 100% tumor take rate in the m.f.p., whereas only 40% of the s.c. injections produced tumors and these occurred several weeks after the appearance of the m.f.p. tumors. Thus, the m.f.p. of nude mice is a favorable site for the growth of human breast carcinomas. MDA-MB-435 tumors produced distant metastases in 80% to 100% of recipients. The most common sites for metastasis were the lymph nodes and lungs, with a lower incidence of metastases in muscle (chest wall and thigh), heart, and brain. New variant cell lines were isolated from metastases in the lungs, brain, and heart. All the cell lines were tumorigenic in the m.f.p., and the lung- and heart-derived metastasis lines produced slightly more lung metastases than the original cell line. However, the brain metastasis variant produced significantly fewer lung metastases. Intravenous inoculation of the spontaneous metastasis-derived cell lines produced few lung colonies. Only cell variants isolated from experimental lung metastases showed enhanced lung colonization potential when reinjected i.v. Our results suggest that the estrogen receptor-negative MDA-MB-435 cell line injected in the m.f.p. of nude mice could be a valuable tool for analysis of the cellular and molecular basis of the metastasis of advanced breast cancer.     

Cultivation of human breast carcinoma in soft agar. Experience with 237 fresh tumour specimens

A total of 237 breast carcinomas have been studied with the Courtenay-Mills (C-M) soft agar method. Cell yields and plating efficiencies (PE) were recorded after various enzyme treatments. The highest cell yields and PEs were obtained with the combination of collagenase 0.5%, hyaluronidase 1000 IE ml-1 and DNase 0.1% and an incubation time of 2 h. Eighty percent of the specimens gave greater than 10 colonies, and 60% formed greater than 30 colonies permitting chemosensitivity studies. The C-M method gave significantly higher PEs than the Hamburger-Salmon (H-S) method. Hormone supplements (insulin, oestradiol, progesterone, hydrocortisone) and also reduced agar concentrations (less than 0.3%) gave marginal stimulation of colony formation. In chemosensitivity studies involving doxorubicin, vincristine and 4-OOH-cyclophosphamide, the C-M method gave dose-response relationships without plateaus.     

Polyamines in relation to human breast, rectal and squamous cell carcinoma

An intimate relation between polyamines and cellular growth is evident from the existing literature; but the role of polyamines in carcinomatous growth has not been established and it remains a controversy whether polyamine analysis can be of any use as a marker for carcinoma. In an attempt to find out the relation of polyamine to carcinomatous tissues, it has been found that putrescine, cadaverine, spermine and spermidine concentrations in breast, rectal and squamous cell carcinomatous tissues, analysed under the present experiment, increased unequivocally. The result has been discussed and a reasonable explanation has been given to support the finding.     

Tumor cell dissemination patterns and metastasis of murine mammary carcinoma

Quantitative studies on the distribution kinetics of isotope-labeled cells from spontaneous murine mammary tumors injected intravenously or arterially showed that cells were rapidly distributed to all organs examined and indicated that the distribution patterns of metastases from such tumors are not primarily determined by the dose of cells delivered to each organ. The preferential colonization of certain organs is therefore considered to depend as much on differential survival and growth of the disseminated tumor cells in unfamiliar metabolic microenvironments, as on vascular sieving effects in organ capillary networks. Further experiments involved transplantation of pieces of nonpulmonary tissue containing trapped mammary tumor cells into syngeneic mice, followed by observation of the animals for several months. From these studies it is concluded that the absence of tumor colonies in extrapulmonary sites after i.v. inoculation is due to their inability to thrive in the organs concerned and not to early death of the original host from heavy pulmonary tumor growth. These results provide further evidence strengthening the conclusion emerging from several independent lines of investigation (Potter et al., Invasion Metastasis, 3: 221-233, 1983; Tarin et al., Cancer Res., 41: 3604-3609, 1981; Tarin et al., Cancer Res., 44: 3584-3592, 1984; Horak et al., J. Natl. Cancer Inst., 76: 913-922, 1986; Nicolson et al., Int. J. Cancer, 38: 289-294, 1986; Naito et al., Invasion Metastasis, 7: 16-29, 1987) that the growth of disseminated tumor cells is inhibited or even abrogated by many of the organs in which the cells sequester after vascular dissemination.