Two different types of concomitant resistance induced by murine tumors: morphological aspects and intrinsic mechanisms.

Concomitant resistance (CR) is the phenomenon according to which a tumor-bearing host inhibits the growth of a secondary implant of the same tumor at a distant site. Confirming and extending previous results of our laboratory, histological studies have revealed that two temporally separate peaks of CR can be detected throughout tumor evolution. The first peak induced by immunogenic small tumors, in euthymic but not in nude mice, is associated with extensive necrosis of the secondary tumor implant and a profuse infiltration of polymorphonuclear granulocytes and mononuclear cells resulting in its final destruction; these features correspond to a typical immunological rejection. The second peak of CR induced by both immunogenic and non-immunogenic large tumors, in euthymic as well as in nude mice, is characterized by a dormant tumor stage with scarce or null mononuclear infiltration, associated with a significant reduction of tumor mitotic index and of the number of PCNA+ cells along with an increase in apoptosis and an arrest in S phase. In previous reports we suggested that a 1000 D serum fraction from mice bearing large tumors could be responsible for the induction of this dormant tumor stage. In this study tumor cells incubated in vitro with that serum factor mimicked the inhibition and cellular alterations observed in vivo in the secondary tumor inhibited by the second peak of CR. Moreover, the passive transfer of this factor by the intra-peritoneal (i.p.) route induced an in vivo inhibition of an i.p. tumor reproducing the image characteristic of the second peak of CR. This represents a direct proof that this serum factor can restrain tumor growth in vivo and that it is, most probably, the effector of the second peak of CR.

Inhibition of metastases by a serum factor associated to concomitant resistance induced by unrelated murine tumors.

Murine lung metatases growing undisturbed by the primary tumor were significantly inhibited by the concomitant resistance induced by a secondary subcutaneous implant of two unrelated tumors. Such inhibition was T-independent since it was also observed in nude mice; its full expression was dependent on the presence of the secondary tumor implant and it was exerted on both macroscopic and microscopic established metastases and not on the process of tumor cell dissemination from the primary tumor. Direct and indirect mechanisms seemed to be involved, the former affecting the metastatic cells per se by causing a decrease in proliferation and an increase in apoptosis while the latter affected neo-vascularization. These antitumor and antiangiogenic effects could be attributed to a serum factor induced by the unrelated tumors generating concomitant resistance. This factor proved to be heat, acid and alkaline resistant and dialysable; it was recovered in an HPLC column with maximum absorption at 215 and 266 nm; it was anionic at neutral pH, exhibiting free carboxil groups and one or more molecules of tyrosine, with a molecular weight between 870 and 1300 Dalton. Intravenous administration of this factor significantly inhibited lung metastases, decreasing mitosis and increasing apoptosis similar to that observed in the presence of the unrelated tumors.

Antiangiogenesis and apoptosis as mediators of concomitant tumor resistance induced by Calu-6, a human lung carcinoma cell line, in nude mice.

Concomitant resistance (CR), the phenomenon by which tumor-bearing hosts are able to inhibit secondary implants of the same tumor at distant sites of the body, has been previously observed by us and others in different murine tumor models. Here, we verified the generation of CR in nude mice by tumors induced by SC inoculation of Calu-6, a human lung carcinoma cell line. Histological analysis of secondary tumors subject to CR did not reveal macrophage infiltration nor cytotoxic signs. Although serum from tumor-bearing mice inhibited in vitro [3H]thymidine uptake by Calu-6 cells, no significant differences in [3H]thymidine labeling index of tumors implanted in the right flank of mice with and without a primary tumor in the left flank were detected. In our model, the presence of a primary tumor hindered remote tumor angiogenesis, as well as serum from tumor-bearing mice inhibited in vitro proliferation of an endothelial cell line derived from a murine hemangioendothelioma. Conversely, an enhancement of the apoptotic index was observed in secondary tumor implants carried out in tumor-bearing mice. The results reported herein show that human tumor cells are capable of inducing CR, and that this phenomenon would be a consequence of an impaired neovascularization as well as an increased programmed cell death at sites distant from the primary tumor.

A serum-mediated mechanism for concomitant resistance shared by immunogenic and non-immunogenic murine tumours.

Resistance of tumour-bearing mice to a second tumour challenge, that is concomitant resistance, was evaluated in euthymic and nude mice using nine tumours with widely different degrees of immunogenicity. Two temporally separate peaks of concomitant resistance were detected during tumour development. The first one was exhibited only by small immunogenic tumours; it was tumour specific and mediated by classical immunological T-cell-dependent mechanisms. The second peak was shared by both immunogenic and non-immunogenic large tumours; it was non-specific, thymus independent and correlated with the activity of a serum factor (neither antibody nor complement) that inhibited the in vitro proliferation of tumour cells. This factor was eluted from a Sephadex G-15 column at fractions corresponding to a molecular weight of approximately 1000 Da and it was recovered from a high-performance liquid chromatography column in one peak presenting maximum absorption at 215 and 266 nm. The data presented in this paper suggest for the first time, to our knowledge, that in spite of the differences between immunogenic and non-immunogenic tumours, a common serum-mediated mechanism seems to underlie the concomitant resistance induced by both types of tumours at late stages of tumour development.

[Role of connective tissue in the tumor-host relationship]. / Rol del tejido conectivo en la relación tumor-huésped.

In the embryo, both differentiation and temporospatial organization are regulated by the mesoderm. Some of these functions are expressed by the connective tissue during wound or organ repair and regeneration. The normal development of the latter depends on the epithelium-mesenchyme interrelationship and the formation of an adequate amount of stroma and a certain type of collagen or proteoglycans. Our hypothesis proposes that cancer is a regenerative process which has failed as a consequence of alterations in the connective tissue. The object of this paper was to investigate whether the connective tissue and the amorphous fundamental substance (SFA) are capable of regulating the proliferation and death of normal and tumor cells and to duplicate the mechanisms involved. The results obtained in vivo, ex-vivo and in vitro experiments indicate that following: 1) SFA exerts a direct and selective cytotoxic effect on tumor cells; 2) SFA reduces the proliferative capacity of normal and tumor cells; 3) both the cytotoxic and antiproliferative effects of SFA are independent of cellular and humoral immune responses but are dependent on the chemical integrity of its component since its denaturalization reduces its antitumoral activity; 4) the tumor cells modulate the regulatory effects of SFA through endocellular enzymes liberated by cell death induced by the cytotoxic action of SFA itself. These results suggest that in the absence of the inhibitory effect of SFA, the tumor cells which remain viable con now proliferate actively due to enzyme stimulation. In conclusion, the regulatory function of the connective tissue on the proliferation and viability of tumor cells would depend on the molecular constitution of SFA.

[Concomitant antitumor resistance]. / Resistencia concomitante antitumoral.

Concomitant resistance of tumor-bearing mice against a second tumor challenge was evaluated in euthymic and athymic mice using 17 tumors with different degrees of immunogenicity. Two temporarily separated peaks of concomitant resistance were detected during tumor development: the first peak was only observed associated with small immunogenic tumors (< 500 m3., it was tumor-specific and mediated by T cell-dependent immunological mechanisms. The second peak was exhibited by large tumors (> 2000 mm3) independently of their immunogenicity; it was non-tumor specific, thymus-independent and correlated with a serum-activity (neither antibodies nor complement) which inhibited the in vitro proliferation of tumor cells. Out of 17 tumors studied, 15 tumors exhibited a moderate or strong concomitant resistance. The remaining two, which exhibited a weak or undetectable concomitant resistance and correlatively, a low or absent serum-inhibitory activity were the only tumors which included lung metastases. This fact suggested a correlation between concomitant resistance, absence of metastases and the existence of an inhibitory factor(s) in the serum. This inhibitory factor was partially characterized: it was resistant to boiling (5-10' at 100 degrees C) and to variations of pH; its molecular weight was estimated between 850 and 1200 D; it was recovered in only one fraction from HPLC (high power liquid chromatography) columns presenting maximum absorption at 215 and 266 nm; amino acid analysis and magnetic nuclear resonance studies suggested the presence of a molecule of thyrosine and one or two molecules of carbohydrates in its structure.

The role of apoptosis in the inhibition of a secondary tumor by concomitant resistance in a mouse model of metastases.

Resistance of tumor-bearing mice to a second tumor challenge, that is, concomitant resistance, was studied using the LB tumor model. In a secondary LB tumor implant inhibited by concomitant resistance an increase in the percentage or apoptotic cells and alterations in cell cycle distribution were observed. Similar alterations were observed in LB tumor cells incubated with serum from tumor-bearing mice. The data presented in this paper suggest that apoptosis is one of the mechanisms involved in tumor dormancy due to concomitant resistance.

Stimulation of angiogenesis as an explanation of Matrigel-enhanced tumorigenicity.

Matrigel, a reconstituted extract of basement membrane, enhances the growth of different human cancer cell lines when transplanted into nude mice. Here that stimulation was confirmed in the BALB/c murine mammary-tumor cell line M3MC, as well as in human colon (SW948) and mammary (MDA-MB-468) carcinoma cell lines transplanted in nude and SCID mice, respectively. Subcutaneous and intra-mammary fat-pad inoculations of Matrigel alone generated an angiogenic response which was macroscopically evident by day 9. Histological analysis of the local host reaction occurring at the site of injection revealed an early peripheral fibroblast response, followed by mononuclear cell infiltration, solid and hollow fibroblast cords projections from the edge to the center of the Matrigel plug, and finally capillary ingrowths. Conditioned media obtained from the gels generated in vivo, acted as very strong chemoattractants for mouse lung capillary endothelial cells, stimulating their motility between 38 and 82 times with respect to the control. Our results suggest an important role of host cells recruited by Matrigel, which could favor angiogenesis of the area and thus facilitate the growth of tumor cells co-inoculated with the basement membrane extract.

Expression of gelatinase/type IV collagenase in tumor necrosis correlates with cell detachment and tumor invasion.

We have previously observed that acellular extracts from necrotic areas (NE) of the non-metastatic murine mammary adenocarcinoma M3, enhance in vitro cell detachment and spontaneous lung metastases. In the present study, using different proteinase inhibitors along with NE, only the calcium chelator EDTA could significantly abrogate the enhanced cell detachment from M3 produced by NE. The typical cleavage products of type IV collagenase were detected inside the tumor necrotic area, mainly in association with necrobiotic cells, as evaluated by Western blot analysis and immunohistochemical assays. Zymography revealed the presence of 72- and 92-kDa gelatinase/type IV collagenase in NE. Moreover, NE increased the in vitro invasive ability of cultured M3 cells. The use of specific antibodies against both 72- and 92-kDa type IV collagenases in the invasion assay showed that only the latter was able to revert the enhanced invasiveness to the baseline. It can be concluded that tumor necrosis is an important source of gelatinase/type IV collagenase, mainly in its 92 kDa form, and plays a major role in tumor invasion.

Correlation between seric antitumor activity and concomitant resistance in mice bearing nonimmunogenic tumors.

Serum from mice bearing five weakly immunogenic or nonimmunogenic tumors inducing concomitant resistance exhibited a growth-inhibitory activity on in vitro proliferation of the tumor cells. This activity was proportional to the intensity of concomitant resistance and correlated with the capacity to restrain metastatic development. It was not attributable to cytotoxic antibodies, was relatively nonspecific, and operated through a cytostatic and reversible mechanism. All attempts to transfer antitumor resistance in vivo by serum inoculation have failed, but this could be attained by parabiosis. Physical and chemical serum treatments suggest that heat-, acid-, and alkali-resistant peptide(s) with molecular weights ranging from 1000 to 3000 could account for this inhibitory effect.

Anti-inflammation induced by counter-irritation or by treatment with non-steroidal agents inhibits the growth of a tumour of non-detected immunogenicity.

Counter-irritation (CI) triggered by different non-specific irritant stimuli delayed the growth of a murine tumour of non-detected immunogenicity. The syngeneic LB tumour transplant by itself also induced CI and decreased the number of leukocytes migrating to a secondary s.c. irritant stimulus, e.g. sponge or carrageenan. On the other hand, partial inhibition of cell migration by treatment with either 0.5 mg kg-1 indomethacin or 0.3 mg kg-1 piroxicam retarded LB tumour growth, presumably by a mechanism unrelated to inhibition of immune responses by PGE2. It is suggested that CI may play a role in the early stages of concomitant resistance.

Tumor necrosis can facilitate the appearance of metastases.

The non-metastatic murine mammary adenocarcinoma M3 and its metastatic variant MM3 were used to evaluate the role of intratumoral necrosis in cell detachment and metastasis. Accelular extracts from necrotic areas of both tumors increased in vitro cellular detachment from M3 but not from MM3 fragments. Furthermore, the in vivo inoculation of the necrotic extracts within non-metastatic M3 tumors gave rise to pulmonary metastases. Histological studies revealed in M3 a central necrosis limited by an uninterrupted peripheral ring of well preserved cells, while in MM3 necrotic and non-necrotic areas alternated. It is concluded that the distribution of necrosis within the primary tumor by facilitating cell detachment is, at least in part, responsible for the development of metastases.

Role of concomitant resistance in the development of murine lung metastases.

An attempt was made to explain the distinct lung metastatic patterns of 2 mammary adenocarcinomas with a common BALB/c origin: M3, which does not induce spontaneous metastases, and MM3 with an almost 100% incidence. No difference between the 2 tumors was detected with respect to host mononuclear cell content, degree of immunogenicity or lung-colony-forming ability. Conversely, there was a marked difference in the capacity to induce concomitant resistance: M3-bearing mice induced stronger and earlier resistance against i.v. challenge of both M3 and MM3 tumor cells than MM3-bearing mice; this resistance was expressed as lower number of lung metastases and lower tumor-cell proliferation in metastatic nodules. M3 was also able to control the development of spontaneous metastases: metastases developed in all M3-excised mice, compared with none in M3-bearing mice, while MM3-bearing mice also bearing a secondary M3 tumor developed fewer metastases than mice bearing MM3 only. This anti-metastatic effect does not appear to depend on classical immunological mechanisms since no difference could be detected between the 2 tumors in response to T cells, NK, macrophages or antibodies.

"Concomitant immunity" in murine tumours of non-detectable immunogenicity.

Various immunization assays were used to demonstrate the lack of immunogenicity of three BALB/c tumours of spontaneous origin and of a fourth one resulting from foreign body tumorigenesis. All four tumours inhibited the growth of a second implant of the same tumour into the contralateral flank. In our tumour models "concomitant immunity" (1) was not mediated by macrophage or T-cell dependent immune reactions: both thymectomized BALB/c and nude mice (treated or untreated with silica) gave the same results as intact mice; (2) showed some degree of non-specificity, inhibiting the growth of a different tumour in 3/4 cases; though, the existence of a specific component could not be discarded; (3) was proportional to the volume of the primary tumour at the time of the second challenge; (4) was dependent on actively growing primary tumour, not being obtained with progressively increasing daily inocula of irradiated tumour cells; (5) was detectable in an actively growing secondary tumour; recurrent growth after partial surgical excision was inhibited and (6) involved cytostasis of the secondary tumour: a syngeneic graft of the overlying skin led to tumour growth while histological studies revealed the presence of viable tumour cells. It is postulated that "concomitant immunity" or resistance can be generated without the active participation of the immune system and that tumour-related factors are, in certain cases, responsible for blocking the growth of secondary tumours.

Macrophages and tumor growth. II. Cell kinetics at the site of allogeneic tumor growth.

The sponge matrix allograft model made possible the growth of an AKR lymphoma in a certain percentage of BALB/c mice making progressors (tumor-bearing) and regressors (tumor-rejecting) simultaneously available. Mice bearing either an AKR kidney allograft or a sponge alone were used as controls. The cell population infiltrating the sponge was evaluated 2, 5, 10, 15, and 21 days after subcutaneous implantation. It consisted of macrophages, lymphocytes, and neutrophils. There was no difference between groups on Day 2 and Day 5. From Day 10 onwards, tumor growth was evident with a clear cut separation between progressors and regressors. The latter behaved like the two control groups except for a significant increase in lymphocytes on Day 21. The progressors showed a striking increase in total cell count from Day 10 onwards. Macrophages were the major population with a maximum value of 201 X 10(6) as compared to 8 X 10(6) in the regressors; their phagocytic and lysosomal activity remained similar in all groups. The lymphocytes showed no variation in absolute numbers but, because of the high cell count in progressors, their ratio to macrophages reached 1:50 on Day 10, making up only 2% of the cell population. Neutrophils were significantly increased in progressors as compared with the other groups. It can be concluded that in the presence of a foreign body reaction a marked influx of macrophages accompanies allogeneic tumor growth.