Maya Ran

The murine Fc-gamma (Fcγ) receptor type II B1 is a tumorigenicity-enhancing factor in polyoma-virus-transformed 3T3 cells

The murine receptor for the Fc portion of IgG is a molecule expressed by cells of the immune system. This study suggests the hypothesis that Fcγ receptor type II B1 (FcγRIIB1) functions as a progression‐enhancing factor when expressed ectopically on non‐lymphoid tumor cells. It has been shown previously that BALB/c 3T3 cells transformed in vitro with polyoma virus (PyV) do not express FcγRII but acquire the expression of this receptor following an in vivo passage in syngeneic mice. The specific FcγRII transcript present in tumor cells was identified in this report as FcγRIIB1 (B1). In order to determine whether or not the ectopically expressed FcγRII plays a role in the progression of these transformed cells, PyV‐transformed 3T3 cells were transfected with B1‐cDNA. The B1 transfected cells were tested for their ability to form local tumors in syngeneic mice, as compared to transfected cells which express the co‐transfecting neomycine resistance (neores) DNA alone or together with the lacZ gene. FcγRIIB1 expressors exhibited a significantly higher tumorigenic phenotype than FcR‐negative controls, though both types of cells exhibited the same growth curve in vitro. The ability of FcγRIIB1 to act as a potentially tumorigenicity‐enhancing factor was also demonstrated as FcγRII was expressed by tumor cells, originating from inoculated FcγRIIB1‐transfected cells, or from inoculation of a mixture of receptor‐positive and ‐negative cells. B1‐expressing cells dominated the tumor‐cell population over non‐expressors. This dominance strengthened the hypothesis that FcR plays a role in tumor progression in vivo.

Contribution of the intracellular domain of murine FC‐gamma receptor type IIB1 to its tumor‐enhancing potential

We have previously shown that Fc gamma receptor type II B1 (FcγRIIB1), when expressed on non‐lymphoid tumor cells, significantly enhanced their tumorigenic phenotype. This study elucidates the role of the intracellular domain of FcγRIIB1 in the enhancement of the malignant phenotype of polyoma‐transformed 3T3 cells. We investigated the tumorigenic potential conferred by different variants of the receptor: FcγRIIB1, a full‐length receptor (B1) whose intracellular region is encoded by exons 8, 9 and 10; FcγRIIB2, a spliced variant (B2) whose cytoplasmic domain comprises exons 9 and 10 and lacks exon 8; and FcγRIIB1‐CT53, a deleted mutant whose cytoplasmic domain contains the fragment encoded by exon 8 alone. We have investigated various properties of cells transfected with each of the above variants: tumorigenicity in syngeneic mice, formation of colonies in soft agar, growth rate, production of soluble receptor and capping of the ligand‐bound receptor. Results show that while the presence of exon 8 did not enhance growth rate in vitro or production of soluble FcγR, it did enhance the tumorigenic phenotype of transfected cells (both in vivo and in vitro growth in soft agar). B1‐expressing cells exhibited a significantly higher tumorigenic phenotype than B2 cells. The presence of exon 8 alone (CT53 mutant) conferred the transfected cells a higher tumorigenic phenotype than FcγR‐negative control cells but lower than intact B1 or B2 cells, indicating that the presence of B1‐specific exon 8 is not sufficient but that the presence of an intact B1 intracellular domain is essential, for conferring the high tumorigenicity phenotype upon cells. We conclude that the capping, following ligand binding contributed by exon 8, and the function contributed by the specific localization of exons 9 and 10 in B1 cells may determine their malignant phenotype.

FcR may function as a progression factor of nonlymphoid tumors

Tumor progression is a multistep process involving genetic and epigenetic changes in a transformed clone. Some of these changes may be induced by host factors which may also select for transformed cellular variants with a high ability to survive and propagate. In this article we review studies showing that receptors for the Fc portion of IgG may be expressed on cells from human or animal tumors of nonlymphoid origin. We also review data demonstrating that at least with respect to cells transformed in vitro with Polyoma virus, transformation per se is not sufficient for the induction of Fc receptor expression. We also summarize preliminary data showing that Fc receptor expression is causally involved in conferring a high malignancy phenotype upon transformed cells. Possible mechanisms to explain these observations are discussed.

Increased expression of Fcγ receptor in cancer patients and tumor bearing mice

In this study we report on some lines of ongoing research performed in our laboratory, in relation to the increased expression of FcR on tumor cells, as well as on cells present in the tumor-bearing host, and its possible role in tumor progression. In a previous study we have shown that a Polyoma virus (PyV)-induced anaplastic carcinoma (SEYF-a tumor) contained an FcR-expressing subpopulation of tumorigenic cells. We tested the effect of in vivo passaging of FcR-expressing and of non-FcR-expressing sub-populations of SEYF-a tumor cells on the expression of FcR, as revealed by the ability of these cells to bind the 2.4G2 monoclonal antibody, which is directed against mouse Fc gamma 2b/gamma 1R. It was found that upon in vivo passaging these two sub-populations became practically identical in their ability to bind anti-Fc gamma R antibody. On the other hand, in vitro passaging of FcR-expressing SEYF-a cells resulted in a gradual decrease in the expression of Fc gamma R. These results, indicating that the expression of Fc gamma R on tumor cells, per se, is dependent on a factor present in the in vivo environment were confirmed using 3T3 cells transformed in vitro by PyV (C) and forming tumors at first injection to mice (CTC). C cultures of various clones did not express Fc gamma R, while CTC cultures (cultures from tumors) became positive. We also detected an increase in the level of a soluble form of Fc gamma 2b/gamma 1R in the circulation of mice bearing PyV induced tumors. This increase paralleled the appearance of palpable tumors. A similar pattern of increase was observed in mice inoculated with the c-H-ras transformed tumorigenic clone 8/F/5, but not in mice inoculated with non-tumorigenic 3T3 cells. Data published by us show that metastatic breast cancer patients had significantly elevated Fc gamma R levels on their peripheral blood mononuclear cells (PBMC). Experiments presented here indicate a direct correlation between increased Fc gamma R levels on PBMC and tumor mass in colon, ovary and lung metastatic carcinoma patients. The possibility that malignantly transformed cells have the potential to cause proliferation of Fc gamma R expressing T cells was tested. It was found that extract derived from r-H-ras transformed 3T3 cells triggers the proliferation of a T cell hybridoma expressing Fc gamma R.

Studies on the Level of Natural Antibodies Reactive with Various Tumor Cells during Urethane Carcinogenesis in BALB/c Mice

Serum from young normal BALB/c mice was found to contain IgM antibodies able to mediate complement-dependent lysis of certain syngeneic or allogeneic tumor target cells. The titer of such naturally occurring antitumor antibodies ( NATA ) was found to increase with aging. A longitudinal serological study comparing the cytotoxicity potential of NATA from normal and from urethan-treated BALB/c mice was performed. It was found that urethan-treated mice that did not develop primary lung-adenomas within the duration of the experiment had significantly lower NATA titers, against one out of 4 target cells assayed, than urethan-treated animals that developed lung adenomas. This difference was evident in two independent experiments. The results suggested that the lower NATA activity of the urethan-treated mice that did not develop tumors existed even before exposure to the carcinogenic insult. This raises the possibility that certain populations could be segregated according to their natural antibody profile into those individuals which will develop primary tumors within a certain period if exposed to a subthreshold amount of carcinogen, and those which will not.

Possibilities of interference with the immune system of tumor bearers by non-lymphoid FcγRII expressing tumor cells

The ectopic expression of Fc gamma RII by PyV transformed 3T3 cells derived from tumors of long latency has been established. It was suggested that this expression is one of several changes conferring upon the cells an increased capacity for survival. We found that in one case cells expressing a very high level of Fc gamma RII had also a very high metastatic phenotype as compared to FcR negative cells. Direct evidence that Fc gamma RIIbl functions as a progression factor was provided by transfection experiments. The transfected gene conferred an increased malignancy and invasive phenotype upon PyV or c-Ha-ras transformed cells. In the present study we tested the possibility that Fc gamma RII expressing tumor cells could interfere with the immune system. The following subjects were investigated: 1) The ability of Fc gamma R on the tumor cells to bind the ligand and/or release IBF. 2) The effect of a local accumulation of ligand and/or IBF (assumed to take place in situ in the tumor) on Fc gamma RII expressing T cells. It was found that both tumor-derived receptor positive and beta l transfected PyV transformed cells were capable of binding aggregated mouse IgG. The binding of bivalent ligand was followed by an increase in membrane Fc gamma RII expression. Also both types of cells were capable of releasing IBF. We then tested the possibility that a local accumulation of IgG within the tumor could effect Fc gamma R expressing T cells. It was found that aggregated mouse IgG (as well as IgGl) could stimulate the proliferation of the T cell hybridoma (T2D4) and other Fc gamma RII expressing T cells. We also found that the expression of beta Fc gamma RII specific mRNA peaked at the logarithmic phase of T2D4 cultures, in parallel with their maximal potential to release IBF. Several pathways for interference with the immune system are suggested.

Phenotypic Properties of 3T3 Cells Transformed in vitro with Polyoma Virus and Passaged Once in Syngeneic Animals

Cloned BALB/c 3T3 cells transformed in vitro with polyoma virus (PyV) acquired a higher tumorigenicity phenotype after a single in vivo passage. Some of the in vivo passaged cells (CTC cells) exhibited also a higher metastatic phenotype than cells from the same clones that were maintained only in culture (C cells). A phenotypic comparison between CTC and C cells was performed. It was found that most CTC lines exhibited a higher binding to laminin compared to their clonal C cell ancestors. Some CTC cells were less sensitive to the cytotoxic effects of TNF-alpha than the corresponding C cells. CTC cells originating from tumors which appeared after a long latency period (late tumors) tended to express Fc gamma RII while CTC cells originating from tumors which appeared after a short latency period (early tumors) as well as the corresponding C cells tended not to express Fc gamma RII. The expression of a membrane epitope recognized by a monoclonal antibody expressing specificity towards PyV transformed cells, was down-regulated on late tumor cells compared to early tumor cells. Transfection of cloned PyV-transformed BALB/c 3T3 cells with the beta 1Fc gamma RII gene augmented the tumorigenicity and metastatic phenotype of the transfectants compared to control transfectants.

Tumor-Localizing Lymphocytotoxic Antibodies

Humoral tumor immunity as expressed within the tumor tissue has been studied in several laboratories, including ours (Witz, 1977). There is little doubt that immunoglobulin molecules are associated with cells lodging in the tumors of humans and of animals. A preferential association between Ig and tumor tissues was indicated in some cases (Ran and Witz, 1970).

Separation of tumor-seeking small lymphocytes and tumor cells using percoll velocity gradients

Using the polyoma virus-induced ascitic SEYF-a tumor, we evaluated isopycnic and velocity sedimentation gradients of Percoll as methods for separating tumor-seeking lymphocytes and tumor cells. It was established that the velocity sedimentation method is suitable for separation of small lymphocytes lodging within the SEYF-a tumor. This was confirmed by a serological analysis of the separated SEYF-a cell population. The results of this study strongly support our previously reported data demonstrating the in vivo coating of the tumor cells proper with potentially cytotoxic antibodies.

Some cellular and molecular characteristics of high and low tumorigenicity variants of polyoma-virus transformed cells

We analyzed several cellular and molecular properties of BALB/c 3T3 cellular clones transformed in vitro with polyoma virus and exhibiting a high or low tumorigenicity phenotype. We also analyzed the same clones after a single in vivo passage in syngeneic mice. This passage invariably induced and/or selected variants exhibiting a very high tumorigenicity phenotype. BALB/c mice bearing tumors induced by the inoculation of the above cells, regardless of their tumorigenicity phenotype, have a lower number of L3T4 positive splenocytes than appropriate controls. The response to Con-A of spleen cells from such mice was also suppressed. Concomitantly, an increase in Mac-1 positive splenocytes could be measured. In spite of the non-specific suppression of T cells, spleen cells from tumor-bearers showed a specific proliferative response to polyoma antigens. Molecular analysis of polyoma transformed cells showed no differences between the various cells with respect to integration of the polyoma viral genes or with respect to src, myc and fos proto-oncogenes. In vitro maintained cells and in vivo passaged cells seemed to differ, however, in the content of polyoma middle T. Whereas polyoma virus transformed cells maintained only in culture never expressed low affinity receptors for IgG (Fc gamma RII), certain in vivo passaged cells did. This expression could be measured both at the protein and the mRNA level. Those in vivo passaged cells which expressed F alpha RII gave tumors following a long latency period. Ongoing experiments will indicate whether or not Fc gamma RII expression is linked to long latency of tumor development.