Organ involvement and phenotypic adhesion profile of 5T2 and 5T33 myeloma cells in the C57BL/KaLwRij mouse.

The aim of this study was to evaluate the tissue infiltration and phenotypic adhesion profile of 5T2 multiple myeloma (MM) and 5T33 MM cells and to correlate it with that observed in human disease. For each line, 30 mice were intravenously inoculated with myeloma cells and at a clear-cut demonstrable serum paraprotein concentration; mice were sacrificed and a number of organs removed. The haematoxylin-eosin stainings on paraffin sections were complemented with immunohistochemistry using monoclonal antibodies developed against the specific MM idiotype. When analysed over time, 5T2 MM cells could be observed in bone marrow samples from week 9 after transfer of the cells. For the 5T33 MM, a simultaneous infiltration was observed in bone marrow, spleen and liver 2 weeks after inoculation. Osteolytic lesions consistently developed in the 5T2 MM, but this was not consistent for 5T33 MM. PCNA staining showed a higher proliferative index for the 5T33 MM cells. The expression of adhesion molecules was analysed by immunohistochemistry on cytosmears: both 5T2 MM and 5T33 MM cells were LFA-1, CD44, VLA-4 and VLA-5 positive. We conclude that both lines have a phenotypic adhesion profile analogous to that of human MM cells. As the 5T2 MM cells are less aggressive than the 5T33 MM cells, their organ distribution is more restricted to the bone marrow and osteolytic lesions are consistently present, the former cell line induces myeloma development similar to the human disease.     

Absence of renal lesions in C57BL/KaLwRij mice with advanced myeloma due to 5T2MM cells

Renal failure is one of the main complications in multiple myeloma (MM) and histopathological lesions are due to light chains accumulation in the kidney. The 5T2MM mouse model closely mimics osteolytic lesions observed in clinics. We studied the occurrence of pathological changes in the kidney of mice inoculated with 5T2MM myeloma cells. No renal lesions due to light chain deposition were observed after histological, immunological staining and dosage of creatinine in serum and urine. PTH levels decreased in 5T2MM mice, confirming the absence of secondary hyperparathyroidism. Osteolytic lesions appear to be the unique consequence of 5T2MM cells inoculation.     

Selective initial in vivo homing pattern of 5T2 multiple myeloma cells in the C57BL/KalwRij mouse

One of the main characteristics of multiple myeloma cells is their predominant localization in the bone marrow. It is, however, unclear whether this is due to a selective initial entry, or whether this entry is more random and other processes like survival and/or growth stimulation, only present in the medullar microenvironment, are unique. To investigate this, in vivo homing kinetics of murine 5T2MM cells shortly after injection were assessed in bone marrow, liver, spleen, lungs, heart, intestines, kidney and testis by tracing of radiolabelled cells, by immunostaining of isolated cells and by polymerase chain reaction analysis. We demonstrated the presence of 5T2MM cells in bone marrow, spleen and liver with all other organs being negative. Adhesion assays of 5T2MM cells to different types of endothelial cells demonstrated a selective adhesion of 5T2MM cells to bone marrow and liver and not to lung endothelial cells. We here demonstrate that the specific in vivo localization of the 5T2MM cells is a result of the combination of a selective entry/adhesion of the 5T2MM cells in the bone marrow, spleen and liver, and a selective survival and growth of these tumour cells in the bone marrow and spleen but not in the liver.     

Endothelial cell-driven regulation of CD9 or motility-related protein-1 expression in multiple myeloma cells within the murine 5T33MM model and myeloma patients.

The cell surface expression of CD9, a glycoprotein of the tetraspanin family influencing several processes including cell motility and metastasis, inversely correlates with progression in several solid tumors. In the present work, we studied the expression and role of CD9 in multiple myeloma (MM) biology using the 5T33MM mouse model. The 5T33MMvitro cells were found to be CD9 negative. Injection of these cells in mice caused upregulation of CD9 expression, while reculturing them resulted in downregulation of CD9. Coculturing of CD9-negative 5T33MMvitro cells with BM endothelial cells (BMECs) resulted in a partial retrieval of CD9. Laser microdissection followed by real-time polymerase chain reaction and immunohistochemistry performed on bone sections of 5T33MMvivo diseased mice demonstrated strong local expression of CD9 on MM cells in contact with BMEC compared to MM cells further away. These findings were also confirmed by immunohistochemistry in MM patients. Neutralizing anti-CD9 antibodies inhibited transendothelial invasion of CD9-expressing human MM5.1 and murine 5T33MMvivo cells. In conclusion, we provide evidence that CD9 expression by the MM cells is upregulated in vivo by close interaction of the cells with BMEC and that CD9 is involved in transendothelial invasion, thus possibly mediating homing and/or spreading of the MM cells.     

The 5T2 mouse multiple myeloma model: characterization of 5T2 cells within the bone marrow

The transplantable C57BL/KaLwRij mouse 5T2 multiple myeloma (MM) is a new animal model for studies on MM in man. Histological examination of the 5T2 MM cells revealed their morphological heterogeneity. In this study we investigated whether this heterogeneity reflects subpopulations of 5T2 MM cells with different biological properties. 5T2 MM bone marrow cells were separated according to their sedimentation velocity (s.v.). When intravenously injected into syngeneic recipient mice, cells with s.v. of 8 mm h-1 led to the development of detectable 5T2 MM after 6 weeks; in contrast, 18 weeks elapsed before the same result was achieved with cells of s.v. lower than 5 mm h-1. Flow cytometric analysis revealed that 5T2 MM cells had an aneuploid DNA content and that most cycling 5T2 MM cells were large, their s.v. rate exceeding 9 mm h-1. It was further demonstrated that about half of all aneuploid cells carried on their membrane the 5T2 MM idiotype. The majority of the idiotype-positive cells had s.v. rate exceeding 6.5 mm h-1 (16%-39%) or lower than 3 mm h-1 (16%-19%). The 5T2 MM was shown to contain subpopulations of cells of different size, proliferation capacity and expression of their membrane 5T2 idiotype; this, most likely reflects cells in different stages of differentiation. The mouse 5T2 MM corresponds also in this respect with MM in man.     

Upregulation of matrix metalloproteinase-9 in murine 5T33 multiple myeloma cells by interaction with bone marrow endothelial cells

MM is a B-cell malignancy mainly characterized by monoclonal expansion of plasma cells in the BM, presence of paraprotein in serum and occurrence of osteolytic bone lesions. MMPs are a family of proteolytic enzymes that can contribute to cancer growth, invasion, angiogenesis, bone degradation and other processes important in the pathogenesis of MM. We investigated MMP-9 production in the 5T33MM murine model. Expression of MMP-9 protein in supernatant and cell extracts was analyzed by gelatin zymography. The in vitro, stroma-independent variant 5T33MMvt showed no protein expression of MMP-9 in contrast to in vivo growing MM cells, 5T33MMvv. However, when 5T33MMvt cells were injected into naive mice and isolated after tumor take (5T33MMvt-vv), they secreted a significant amount of MMP-9. These results were confirmed by specific staining of cytospins with an anti-MMP-9 antibody. The MMP-9 production by 5T33MMvt-vv cells disappeared when the cells were recultured in vitro. These data demonstrated that upregulation of MMP-9 occurs in vivo and that this process is dependent on the microenvironment. Cocultures of 5T33MMvt cells with STR10 BMECs induced MMP-9 in MM cells, as determined by both gelatin zymography and flow-cytometric analysis. In conclusion, our results demonstrate that MMP-9 production by MM cells is upregulated in vivo by the interaction of MM cells with BMECs.     

IFN-α1 gene transfection completely abolishes the tumorigenicity of murine B16 melanoma cells in allogeneic DBA/2 mice and decreases their tumorigenicity in syngeneic C57BL/6 mice

The murine B16 melanoma (H-2^b) was transfected with a retroviral vector containing the mouse IFN-α₁ gene. IFN-α₁-transfected cells produced IFN-α in vitro and exhibited an altered phenotype characterized by a decreased rate of multiplication, enhanced expression of H-2 antigens, an antiviral state to VSV, and decreased pigmentation. Control and IFN-α₁-transfected cells were tested for their ability to grow in syngeneic (H-2^b) CS7B1/6 and allogeneic (H-2^d) DBA/2 mice. IFN-α₁producing B16 clones were less tumorigenic after s.c, i.p., and i.v. routes of injection than IFN-non-producer BI6 clones in syngeneic CS7B1/6 mice. IFN-α₁-producing B16 cells were, however, totally rejected by allogeneic DBA/2 mice regardless of the routes and inocula tested, while control B16 cells grew in and killed DBA/2 mice. The total rejection of IFN-α₁-transfected B16 cells in allogeneic mice appeared to be dependent on T cells as these cells grew in DBA/2 nude mice. Incubation of IFN-α-producing clones with anti-mouse IFN-α/β prior to injection into C57BI/6 mice did not enhance their tumorigenicity. Likewise, injection of C57BI/6 and DBA/2 mice with antibody to IFN-α/β did not enhance the tumorigenicity of IFN-α₁-transfected cells. C57B1/6 mice immunized with irradiated IFN-α₁ cells were only slightly protected against a subsequent challenge with parental B16 cells. In contrast, DBA/2 mice immunized with irradiated IFN-α₁ cells exhibited tumor-specific, long-lasting immunity to subsequent challenge with parental B16 cells. © 1995 Wiley-Liss, Inc.     

The 5T mouse multiple myeloma model: absence of c-myc oncogene rearrangement in early transplant generations

Consistent chromosomal translocations involving the c-myc cellular oncogene and one of the three immunoglobin loci are typical for human Burkitt's lymphoma, induced mouse plasmacytoma (MPC) and spontaneously arising rat immunocytoma (RIC). Another plasma cell malignancy, multiple myeloma (MM), arising spontaneously in the ageing C57BL/KaLwRij mice, was investigated in order to see whether the MM cells contain c-myc abnormalities of the MPC or RIC type. Rearrangement of the c-myc oncogene was found in the bone marrow cells only in 5T2 MM transplantation line in a mouse of the 24th generation and in none of the seven other MM of the 5T series which were of earlier generations. Since the mouse 5T MM resembles the human MM very closely, including the absence of consistent structural c-myc oncogene abnormalities, it can serve as a useful experimental model for studies on the aetiopathogenesis of this disease.     

Intratumoral immunization with tumor RNA-pulsed dendritic cells confers antitumor immunity in a C57BL/6 pancreatic murine tumor model

Pancreatic tumors lack adequate recruitment of immunocompetent cells, especially dendritic cells (DCs). We have shown previously that coculturing of natural killer-like T cells with DCs transfected with pancreatic tumor cell line-derived RNA reverses pancreatic carcinoma cell resistance by directly triggering natural killer-like T lymphocytes in vitro. In the present study, we tested triggering of specific T lymphocytes in vivo by using an immunocompetent mouse strain (C57BL/6). Syngenic, bone marrow-derived DCs were pulsed with tumor RNA derived from the pancreatic cell line PANC02. This cell line is a ductal pancreatic adenocarcinoma and shows high resistance to every known class of clinically active antitumor agent. PANC02 cells were implanted orthotopically via ultrasound guidance and led to pancreatic tumor formation in all of the mice. Thereafter, tumor RNA-pulsed DCs were injected intratumorally. Intratumoral administration of tumor RNA-pulsed DCs induced significantly more potent protective immunity than s.c. or i.v. administration. It was significantly more effective than administration with unpulsed DCs or DCs pulsed with a control tumor RNA derived from a lymphomatous cell line (EL4). The antitumor effect was caused by induction of antigen-specific T lymphocytes as shown by additional in vitro studies. These results favor intratumoral injection of tumor RNA-pulsed DCs for immunotherapy of pancreatic cancer.     

Spontaneous and induced preleukemia cells in C57BL/6 mice:brief communication.

A transplantation bioassay method was used to verify the presence of preleukemia cells in C57BL/6 mice shortly after leukemogenic treatment or in relation to age increase. Preleukemia cells were identified mainly among bone marrow cells of old C57BL/6 mice or within 10 to 30 days after leukemogenic treatment of young mice with radiation-induced leukemia virus variants, fractionated doses of irradiation, or 7,12-dimethylbenz[a]anthracene (DMBA), although the overt disease did not occur until many months later. Mice could carry preleukemia cells without necessarily developing overt leukemia. Since the leukemogenic agents used in the present studies induced T-leukemias, the role of the thymus in the induction of preleukemia cells was tested. Thymectomy affected viral transformation but did not diminish the number of preleukemia cells induced by DMBA or X-ray.     

Src kinase induces tumor formation in the c-SRC C57BL/6 mouse

Src kinase has been linked as a causative agent in the progression of a number of cancers including colon, breast, lung and melanoma. Src protein and activity levels are increased in colorectal cancer and liver metastases arising secondary to colon cancer. However, although Src protein is increased in colon cancer as early as the adenomatous polyp stage, a role for Src in carcinogenesis has not been established. We developed the c-SRC transgenic mouse in the C57BL/6 strain to address the issue of carcinogenesis in cells with high levels of Src expression. The transgene was constructed with the human c-SRC gene downstream of the mouse metallothionein promoter to create zinc inducible gene expression. In these C57BL/6 mice, Src protein was increased in a number of tissues both with and without zinc induction. No additional carcinogenic agent was administered. After 20 months, mice were assessed for tumor development in the liver and GI tract, as well as other organs. Of the mice with the transgene, 15% developed tumors in the liver while no tumors were detected in wild type C57BL/6 mice. A further study was conducted by crossing c-SRC C57BL/6 mice with p21 nullizygous mice to determine the effect of oncogene expression combined with inactivation of the tumor suppressor gene, p21. Addition of the c-SRC transgene to the p21-/- background increased tumor formation almost 3-fold, while it increased metastasis 6-fold. The data from our study show, for the first time, that Src kinase may play a role in carcinogenesis.     

1,2-Dimethylhydrazine-induced nuclear aberrations in A/J and C57BL/6J mouse colonic crypts

A single exposure to 1,2-dimethylhydrazine [(DMH) CAS: 540-73-8] produces several forms of aberrant nuclei in the crypts of the murine colon. The frequency of nuclear aberrations (NAs) was examined in the distal colonic crypts in DMH-sensitive A/J mice and relatively DMH-resistant C57BL/6J mice before and after a single exposure to DMH. NAs, mitotic figures, and crypt column heights were scored for all animals as a function of time following administration of DMH. In both strains there was a significant increase in the absolute and relative frequency of NAs by 12 hours, with a corresponding drop and subsequent overshoot in the mitotic index by 48 hours after DMH. The temporal changes in crypt column height correlate closely with the temporal changes in frequency of NAs in both strains. The results showed that both inbred strains respond to acute DMH exposure in a similar and parallel fashion over time. It was concluded that the NA index assay is a sensitive method for detecting early DMH exposure. However, this assay does not relate to ultimate outcome after chronic DMH exposure and should not be used as a predictor of eventual neoplastic transformation of colonic mucosa with this carcinogen.     

Ochratoxin A carcinogenesis in the (C57BL/6J X C3H)F1 mouse

The potential carcinogenic effects of the mycotoxin ochratoxin A [(OA); CAS: 303-47-9] were assessed in a 24-month feeding study in male and female (C57BL/6J X C3H)F1 (B6C3F1) mice. The mice were assigned to 3 groups of 50 males and 50 females each; group 1 mice were the controls, group 2 mice were fed 1 ppm OA, and group 3 mice were fed 40 ppm OA. Renal neoplasms, both carcinomas and adenomas, were found only in male mice of the 40-ppm dose group. Fourteen of 49 animals that survived at least 20 months had neoplasms morphologically consistent with renal carcinoma. Renal adenomas were present in some of these mice and in other 40-ppm-group males, making a total of 26 mice with renal adenomas. All male mice of the 40-ppm dose group had nephropathy characterized by varying degrees of renal tubular dilation, attenuation and hyperplasia of lining epithelium, and proliferation of regenerative tubules. Females of the 40-ppm dose group had similar but less severe renal changes but no carcinomas or adenomas. Compound-related renal lesions were absent in the 1-ppm dose group. The incidence of hepatocellular neoplasms was slightly increased in male and female mice fed diets containing OA. These results indicate that OA is a renal carcinogen in male B6C3F1 mice and a hepatic carcinogen in female mice of this strain.     

Murine 5T multiple myeloma cells induce angiogenesis in vitro and in vivo

Multiple myeloma is a B cell malignancy. Recently, it has been demonstrated that bone marrow samples of patients with multiple myeloma display an enhanced angiogenesis. The mechanisms involved seem to be multiple and complex. We here demonstrate that the murine 5T multiple myeloma models are able to induce angiogenesis in vitro by using a rat aortic ring assay and in vivo by determining the microvessel density. The rat aortic rings cultured in 5T multiple myeloma conditioned medium exhibit a higher number of longer and more branched microvessels than the rings cultured in control medium. In bone marrow samples from 5T multiple myeloma diseased mice, a statistically significant increase of the microvessel density was observed when compared to bone marrow samples from age-matched controls. The angiogenic phenotype of both 5T multiple myeloma cells could be related, at least in part, to their capacity to produce vascular endothelial growth factor. These data clearly demonstrate that the 5T multiple myeloma models are good models to study angiogenesis in multiple myeloma and will allow to unravel the mechanisms of neovascularisation, as well as to test new putative inhibitors of angiogenesis.     

p53 mutations in C57BL/6J murine thymic lymphomas induced by gamma-irradiation and N-methylnitrosourea.

Genomic DNA from thymus tissue obtained from 47 C57BL/6J animals treated with the DNA alkylating agent N-methylnitrosourea or gamma-irradiation were screened for the presence of p53 mutations by using the single strand conformation polymorphism assay. Mutations were detected in 13% (4 of 30) of primary thymic lymphomas but none of 17 early stage lymphomas. The frequency of p53 mutations was the same in tumors induced by N-methylnitrosourea (2 of 15) or by gamma-irradiation (2 of 15). Mutations occurred in the highly conserved regions of the p53 gene in exons 5, 7, and 8. G:C to A:T transitions were commonly observed. One of 4 of the tumors analyzed contained two p53 mutations in exons 7 and 8. A previous study of the same tumors showed that ras mutations occurred with high frequency (greater than 50%) (E. W. Newcomb et al., Cancer Res., 48:5514-5521, 1988). Our data suggest that p53 mutations do not play a major role in carcinogen-induced thymic lymphomas studied here.     

Degradation of cell-bound antibodies by tumor cells from C57BL/6 mice.

A kinetic study of the disappearance of sensitizing antibody from metabolizing EL 4 cells in C57BL/6 mice revealed that this disappearance was at least partly due to the degradation of the antibody into dialyzable products. The degradation took place at 37 degrees C, but not at 4 degrees C, and occurred on or inside the cell. The process was specific inasmuch as only those IgG molecules that could bind to the cells were degraded, whereas unrelated third-party antibodies present in the culture medium remained intact. Although nonmalignant cells also have the capacity to degrade cell-bound antibodies, possibly the degradation of proteinaceous structures on antitumor effector mechanisms is, in part, responsible for decreased antitumor immunity.