Effect of monoclonal antibodies to early pregnancy factor (EPF) on the in vivo growth of transplantable murine tumours

Summary Neutralisation studies with monoclonal antibodies (mAbs) specific for early pregnancy factor (EPF) have shown it to be essential for the continuation of pregnancy in mice and the growth of some tumour cells in vitro. These studies report that the mAbs are also able to limit the growth of two murine tumour lines transplanted s. c. The development of MCA-2 tumours in CBA mice was unaffected by the injection of 1 mg anti-EPF IgM at the time of tumour cell inoculation. However, four doses of 500 µg anti-EPF, injected one dose per day for 4 days after tumour cell inoculation, significantly retarded tumour development such that no tumours were palpable on day 13. A similar dose regimen of control IgM had no effect on tumour size. Dose/response studies revealed that lower doses of anti-EPF administered after tumour cell inoculation were effective in retarding the growth of the MCA-2 tumours. The effect of anti-EPF mAb administration on the growth rate of palpable B16 tumours established s. c. in C57BL/6 mice was also determined. Tumours injected with 6 mg anti-EPF 5/341 or anti-EPF 5/333 mAbs showed significant decrease in the uptake of [³H]thymidine into tumour tissue, measured 16 h after injection. Furthermore, titration of sera for active EPF showed that a significant reduction in the EPF titre was associated with a significant inhibition of tumour DNA synthesis. Thus it appears that neutralisation of EPF retards tumour growth both in vitro and in vivo. In vitro the effects must be due to anti-EPF mAb interfering with a direct mechanism that contributes to the maintenance of cells in the active growing phase. However, in vivo host immunological mechanism that are modified to allow tumour survival may also be affected. The presence of EPF-induced suppressor factors curculating in the serum of tumour-bearing mice has been confirmed and the contribution of such factors to tumour progression must now be investigated.     

Relationship between the effectiveness of the antitumor action of sarcolysine and the latency period between transplantation and tumor appearance

Sarcoma induced with 3-methylcholantrene in mice was transplanted to syngeneic recipients. The latent period of the tumour growth varied from 7 to 26 days. Sarcolysine was injected once when the tumour became palpable. When the tumours were revealed 7 to 8 and 10 to 15 days after the inoculation, the tumour growth inhibition was accompanied by increased survival of mice, particularly marked at the latter period. When the tumours were revealed 17 to 20 or 22 to 26 days after the transplantation, inhibition of the tumour growth was not accompanied by any increase of survival.     

Inhibition of growth and spontaneous metastasis of syngeneic transplantable tumors by an aromatic retinoic acid analogue

Summary Aromatic retinoic acid analogues selected for their favourable therapeutic ratios were tested for their effects on the growth (in vivo and in vitro) and spontaneous metastasis of a variety of murine sarcomas and carcinomas. Ro 10-1670 (a trimethylmethoxyphenyl analogue of retinoic acid) was used in in vitro studies, while its corresponding ethyl ester Ro 10-9359 was used for oral administration. Four of six fibrosarcomas, one of three squamous cell carcinomas, and one of five mammary adenocarcinomas responded to retinoid treatment in vivo by reduced growth rates (first detectable after 8–10 days), and in some cases by complete regression. The magnitude of the response was directly proportional to tumour immunogenicity, and eight tumours which failed to respond to retinoids did not evoke detectable transplantation immunity in syngeneic recipients. Retinoid administration did not significantly inhibit the development of spontaneous metastasis of non-immunogenic tumours, but decreased the incidence of secondary disease in the case of tumours of moderate immunogenicity. In vitro, retinoid treatments were generally without significant effects on tumour cell growth rate or morphology, and where growth inhibition was obtained it did not correlate with in vivo tumour responsiveness. No evidence of increased differentiation of retinoid-treated tumours was obtained either in vitro or in vivo. Taken together, the data suggest that in the 14 transplantable syngeneic tumours studied the inhibitory effects of retinoids on tumour growth and metastasis in vivo were mediated indirectly by potentiation of cell-mediated immunity directed against antigenic determinants on the tumour cell surface.     

Inhibition of c‐MET increases the antitumour activity of PARP inhibitors in gastric cancer models

Gastric cancer is the fifth most common malignancy and the third leading cause of cancer‐related death worldwide. Activation of c‐MET increases tumour cell survival through the initiation of the DNA damage repair pathway. PARP is an essential key in the DNA damage repair pathway. The primary role of PARP is to detect and initiate an immediate cellular response to single‐strand DNA breaks. Tumours suppressor genes such as BRCA1/2 are closely associated with the DNA repair pathway. In BRCA1/2 mutations or deficiency status, cells are more likely to develop additional genetic alterations and chromosomal instability and can lead to cancer. In this study, we investigate the role of c‐MET and PARP inhibition in a gastric cancer model. We exploited functional in vitro and in vivo experiments to assess the antitumour potential of co‐inhibition of c‐MET (SU11274) and PARP (NU1025). This leads to a reduction of gastric cancer cells viability, especially after knockdown of BRCA1/2 through apoptosis and induction of γ‐Η2ΑΧ. Moreover, in AGS xenograft models, the combinatorial treatment of NU1025 plus SU11274 reduced tumour growth and triggers apoptosis. Collectively, our data may represent a new therapeutic approach for GC thought co‐inhibition of c‐MET and PARP, especially for patients with BRCA1/2 deficiency tumours.     

In situ vaccination against a non-immunogenic tumour using intratumoural injections of liposomal interleukin 2

Cancers appear to escape surveillance by the immune system at least in part because they fail to induce a protective immune response. Therapeutic vaccines based on specific tumour antigens and tumour cells modified ex vivo by genetic techniques are but two strategies being used to circumvent this problem. In this report, we describe a simple, yet effective alternative in which tumour-specific responses are induced by in situ administration of a well-characterized liposomal formulation of the cytokine interleukin 2 (IL-2). Using the non-immunogenic B16 melanoma model, intratumoural injections of liposomal IL-2 L(IL2), were shown to induce a long-lived immune response specific for the injected tumour. In conjunction with subsequent removal of the primary tumours by surgery, the injections increased mean survival to 57 days from a control value of 32 days and partially protected surviving mice against re-challenge with B16. L(IL2) induced an early infiltration of inflammatory cells within the tumours which was followed several days later by an influx of CD3+ T cells. The cellular influx and a coincident decrease in tumour growth were noted in both injected tumours and a second non-injected tumour on the same animal, thereby demonstrating the systemic nature of the immune response. Intratumoural injections of soluble IL-2 at the same dose failed to induce B16-specific cellular immunity or to prolong survival of the mice. Thus, liposomal formulation of the cytokine was fundamental to successful induction of immunity by this in situ vaccination regimen.     

Regulation of Ia+ reticulum cell sarcoma (RCS) growth in syngeneic SJL/J mice. I. Inhibition of tumor growth by passive administration of L3T4 monoclonal antibody before or after tumor inoculation

Spontaneously arising reticulum cell sarcoma (RCS) tumors in SJL/J mice stimulate syngeneic host T lymphocytes to proliferate and are dependent on host T cells for maintenance and growth. Tumor-associated Ia antigens have been implicated in the proliferative response both in vivo and in vitro, and the responding T cells are predominantly Lyt-1+2- L3T4+. We hypothesized that elimination or depletion of the responding L3T4 subpopulation in vivo should inhibit growth of transplantable RCS tumors, and continued RCS growth may be dependent on the continued presence of L3T4 cells. This hypothesis was tested experimentally by examining the effect of passive administration of L3T4 monoclonal antibody (mAb) into SJL/J mice either before or at different times after tumor inoculation. The tumor inoculum used killed all mice 15 to 30 days after injection. Administration of a single dose of L3T4 mAb 4 days before tumor inoculation resulted in complete depletion of L3T4 cells and complete inhibition of tumor growth. The antibody-treated mice survived with no sign of tumor growth even after complete recovery of L3T4+ cells. These results demonstrate that initiation of tumor growth is dependent on host L3T4+ cells. Administration of mAb as late as 7 days after tumor inoculation resulted in inhibition of tumor growth, and administration of mAb at day 10 resulted in significant inhibition of tumor growth. Compared with the kinetics of tumor growth in normal control mice, administration of L3T4 after tumor inoculation results in tumor growth arrest. These findings demonstrate that continued tumor growth in vivo is dependent on the presence of L3T4+ cells. In the RCS system, the present studies show that administration of mAb to L3T4+ cells is therapeutic in that it inhibits the induction of tumor growth, and it also prevents tumor growth in tumor-bearing animals.     

α‐Mangostin suppresses the de novo lipogenesis and enhances the chemotherapeutic response to gemcitabine in gallbladder carcinoma cells via targeting the AMPK/SREBP1 cascades

High rates of de novo lipid synthesis have been discovered in certain kinds of tumours, including gallbladder cancer (GBC). Unlike several other tumours, GBC is highly insensitive to standard adjuvant therapy, which makes its treatment even more challenging. Although several potential targets and signalling pathways underlying GBC chemoresistance have been revealed, the precise mechanisms are still elusive. In this study, we found that α‐Mangostin, as a dietary xanthone, repressed the proliferation and clone formation ability, induced cell cycle arrest and the apoptosis, and suppressed de novo lipogenesis of gallbladder cancer cells. The underlying mechanisms might involve the activation of AMPK and, therefore, the suppression of SREBP1 nuclear translocation to blunt de novo lipogenesis. Furthermore, SREBP1 silencing by siRNA or α‐mangostin enhanced the sensitivity of gemcitabine in gallbladder cancer cells. In vivo studies also displayed that MA or gemcitabine administration to nude mice harbouring NOZ tumours can reduce tumour growth, and moreover, MA administration can significantly potentiate gemcitabine‐induced inhibition of tumour growth. Corroborating in vitro findings, tumours from mice treated with MA or gemcitabine alone showed decreased levels of proliferation with reduced Ki‐67 expression and elevated apoptosis confirmed by TUNEL staining, furthermore, the proliferation inhibition and apoptosis up‐regulation were obviously observed in MA combined with gemcitabine treatment group. Therefore, inhibiting de novo lipogenesis via targeting the AMPK/SREBP1 signalling by MA might provide insights into a potential strategy for sensitizing GBC cells to chemotherapy.     

Biofilm formation by Salmonella enterica serovar Typhimurium colonizing solid tumours

Systemic administration of Salmonella enterica serovar Typhimurium to tumour bearing mice results in preferential colonization of the tumours and retardation of tumour growth. Although the bacteria are able to invade the tumour cells in vitro, in tumours they were never detected intracellularly. Ultrastructural analysis of Salmonella-colonized tumours revealed that the bacteria had formed biofilms. Interestingly, depletion of neutrophilic granulocytes drastically reduced biofilm formation. Obviously, bacteria form biofilms in response to the immune reactions of the host. Importantly, we tested Salmonella mutants that were no longer able to form biofilms by deleting central regulators of biofilm formation. Such bacteria could be observed intracellularly in immune cells of the host or in tumour cells. Thus, tumour colonizing S. typhimurium might form biofilms as protection against phagocytosis. Since other bacteria are behaving similarly, solid murine tumours might represent a unique model to study biofilm formation in vivo.     

Study of cytokines involved in the prevention of a murine experimental breast cancer by kefir

Previous studies have shown that compounds released during milk fermentation by Lactobacillus helveticus are implicated in the antitumour effect of this product. Here the effects of the consumption, during 2 or 7 days, of kefir or kefir cell-free fraction (KF) on the systemic and local immune responses in mammary glands and tumours using a murine hormone-dependent breast cancer model were studied. In the tumour control group, mice did not receive these products. At the end of the feeding period, mice were injected subcutaneously with tumour cells in the mammary gland. Four days post-injection, they received kefir or KF on a cyclical basis. Rate of tumour development, cytokines in serum; mammary gland tissue, and tumour isolated cells were monitored. Two-day cyclical administration of both products delayed tumour growth. Both kefir and KF increased IL-10 in serum and decreased IL-6(+) cells (cytokine involved in oestrogen synthesis) in mammary glands. Two-day cyclical administration of KF increased IL-10(+) cells in mammary glands and in tumours and decreased IL-6(+) cells in tumour. This study demonstrated the modulatory capacity of KF on the immune response in mammary glands and tumours and the importance of the administration period to obtain this effect.     

Dietary mistletoe lectin supplementation and reduced growth of a murine non-Hodgkin lymphoma

The growth of a murine non-Hodgkin lymphoma (NHL) tumour has been shown to be reduced by incorporating mistletoe lectin (ML-1) into the diet. The morphological characteristics of NHL tumours in mice fed ML-1-supplemented diets were different from those in LA (control)-fed mice. The degree of mitotic activity was lower and nuclear area reduced. The degree of lymphocyte infiltration was increased in tumours from ML-1 fed mice and this was accompanied by a high incidence of apoptotic bodies. Visual observation of NHL tumours from individuals fed ML-1 diet showed a poorly developed blood supply in contrast to control-fed mice. A major reduction in number of blood capillaries in NHL tumours was confirmed by microscopic evaluation of tumour sections. The results suggested an anti-angiogenic response in ML-1-fed mice. The feeding of ML-1 compared to control diet thus provided several identifiable changes in the morphology of NHL tumours which were consistent with the observed reduction in tumour weight. There was no longer histological evidence of viable tumour in 25% mice fed the ML-1 diet for 11 days. Morphological studies of the small bowel indicated (a) that the lectin induces hyperplasia, and (b) that the lectin binds avidly to lymphoid tissue of Peyer's patches. There was evidence of limited endocytosis of the lectin. An experiment where ML-3 was added to the diet of mice three days after inoculation of tumour cells showed that the lectin was able to slow down further growth of an established tumour. The results show that ML lectins induce powerful anti-cancer effects when provided by the oral route.     

Active Immunotherapy with Corynebacterium parvum and Chemotherapy in Murine Fibrosarcomas

Corynebacterium parvum used alone to enhance immunological reactivity produced transient inhibition of the growth of chemically induced isogenic mouse tumours. Attempts were made to combine C. Parvum with cyclophosphamide to see whether this would increase the latter''s effectiveness in inhibiting early but established tumours. Of the various regimens tested, the administration of the C. parvum 12 days after a single dose of chemotherapy produced dramatic inhibition of tumour growth and resulted in complete and lasting regressions in up to 70% of tumour-bearing animals. The most important variable in this regimen is the time between the chemotherapy and the subsequent immunotherapy.It is possible that non-specific active immunotherapy with agents such as C. parvum may be a valuable adjunct to the conventional cyto-reductive treatments of cancer, but the time of administration of such therapy is probably critical for each tumour and for each chemotherapeutic regimen.     

Cancer growth, response to treatment and survival time in mice: Beneficial effect of the prostaglandin synthesis inhibitor flurbiprofen

Adult mice were injected subcutaneously with cells from a syngeneic metastasizing mammary cancer. Daily treatment with flurbiprofen starting before injection of the cancer cells reduced tumour growth and lengthened the survival of mice whose tumours were excised at 3 weeks.When low doses of radiotherapy and chemotherapy were given, additional treatment with flurbiprofen starting 25 days after injecting the cancer cells substantially inhibited tumour growth.     

Oral Tolerance to Cancer Can Be Abrogated by T Regulatory Cell Inhibition

Oral administration of tumour cells induces an immune hypo-responsiveness known as oral tolerance. We have previously shown that oral tolerance to a cancer is tumour antigen specific, non-cross-reactive and confers a tumour growth advantage. We investigated the utilisation of regulatory T cell (Treg) depletion on oral tolerance to a cancer and its ability to control tumour growth. Balb/C mice were gavage fed homogenised tumour tissue – JBS fibrosarcoma (to induce oral tolerance to a cancer), or PBS as control. Growth of subcutaneous JBS tumours were measured; splenic tissue excised and flow cytometry used to quantify and compare systemic Tregs and T effector (Teff) cell populations. Prior to and/or following tumour feeding, mice were intraperitoneally administered anti-CD25, to inactivate systemic Tregs, or given isotype antibody as a control. Mice which were orally tolerised prior to subcutaneous tumour induction, displayed significantly higher systemic Treg levels (14% vs 6%) and faster tumour growth rates than controls (p<0.05). Complete regression of tumours were only seen after Treg inactivation and occurred in all groups - this was not inhibited by tumour feeding. The cure rates for Treg inactivation were 60% during tolerisation, 75% during tumour growth and 100% during inactivation for both tolerisation and tumour growth. Depletion of Tregs gave rise to an increased number of Teff cells. Treg depletion post-tolerisation and post-tumour induction led to the complete regression of all tumours on tumour bearing mice. Oral administration of tumour tissue, confers a tumour growth advantage and is accompanied by an increase in systemic Treg levels. The administration of anti-CD25 Ab decreased Treg numbers and caused an increase in Teffs. Most notably Treg cell inhibition overcame established oral tolerance with consequent tumor regression, especially relevant to foregut cancers where oral tolerance is likely to be induced by the shedding of tumour tissue into the gut.     

Cancer growth, response to treatment and survival time in mice: beneficial effect of the prostaglandin synthesis inhibitor flurbiprofen.

Adult mice were injected subcutaneously with cells from a syngeneic metastasizing mammary cancer. Daily treatment with flurbiprofen starting before injection of the cancer cells reduced tumour growth and lengthened the survival of mice whose tumours were excised at 3 weeks. When low doses of radiotherapy and chemotherapy were given, additional treatment with flurbiprofen starting 25 days after injecting the cancer cells substantially inhibited tumour growth.     

Photoinduced antitumour effect of hypericin can be enhanced by fractionated dosing

The in vivo antitumour activity of the natural photosensitizer hypericin was evaluated. C3H/DiSn mice were inoculated with fibrosarcoma G5:1:13 cells. When the tumour reached a volume of 40-80mm3 the mice were intraperitoneally injected with hypericin, either in a single dose (5mg/kg; 1 or 6h before laser irradiation) or two fractionated doses (2.5 mg/kg; 6 and 1 h before irradiation with laser light; 532 nm, 70mW/cm2, 168 J/cm2). All tumours in control groups treated with hypericin alone as well as those irradiated with laser light alone had similar growth rates and none of these tumours regressed spontaneously. Complete remission of tumour in photodynamic therapy (PDT)-treated groups was similar (14-17% single dose vs. 33% fractionated dose), but the fractionated schedule of hypericin dosing was found to be more efficient than the single dose, measured by survival assay (p < 0.05). Our experimental model showed that fractionated administration of hypericin can produce a better therapeutic response than single administration.     

Age-related changes in the cell proliferation of ATPC+ mouse ascites tumour

The growth of ATPC+, an ascites tumour derived from a spontaneous mammary carcinoma in BALB/c+ mice, was studied at different ages. It was observed that the number of cells increases rapidly during the first 5 days after implantation. Thereafter, the cell number increases more slowly, reaching a plateau after 8 days. This slowing-down is not due to a reduction in the growth fraction but to a lengthening of the cell cycle. Between 5 and 8 days the duration of all phases increases, including the S phase, which increases from 5.2 h in 5-day tumours to 8.2 h in 8-day tumours. In 12-day tumours both the cell cycle and S phase are only slightly longer than in 8-day tumours whereas the growth fraction is reduced. The slowing-down of cell growth can be attributed to growth fraction reduction rather than cell loss, which is maximal in the 5-day tumour. At this age the time course of the percent labelled cells and of the number of grains/nucleus suggests reutilization of [3H]-thymidine. Incorporation of [3H]-thymidine/cell decreases sharply in 12-day tumours due to a reduced availability of thymidine, which is degraded to thymine in the in vivo ascitic fluid faster than in 8-day tumours. This indicates an age-related change in the ascitic fluid composition.     

Effect of local interleukin-2 treatment on spontaneous tumours of different immunogenic strength

Transplantable tumour lines established from spontaneous tumours of BALB/c, CBA, and DBA/2 mice displayed different immunogenic strength. This report describes tumour susceptibility to interleukin-2 (IL-2) therapy in relation to tumour immunogenicity. The following tumour lines were used: X5, X6, and X9 mammary tumours of DBA/2, BALB/c, and CBA origin respectively, X7 carcinoma of BALB/c and X18 papilloma of CBA mice. Two spontaneous tumours of long transplantation history, SL2 lymphoma (SL2) of DBA/2 and Madison lung carcinoma M109 (M109) of BALB/c origin, were used as control systems. Experimental mice were transplanted with different inocula of tumour cells at day 0; treatment with IL-2 was initiated on days 1–3 or delayed until day 10 and consisted of daily injections of low doses of 5000 or 20 000 U/mouse given five times a week for a period of 3 weeks. Treatment of SL2 (2 × 10⁴ cells injected i.p.) consisted of i.p. injections of 5000 or 20 000 U IL-2/mouse given on days 10–14 after tumour transplantation. IL-2 therapy of SL2-bearing DBA/2JIco mice resulted in a significant proportion of cures; however, no response to IL-2 treatment was achieved in SL2-bearing DBA/2CrIiw mice. BALB/c mice with the i.p. transplant of M109 responded to IL-2 treatment with 40% increase in lifespan. The low-dose IL-2 therapy of the five spontaneous tumours resulted, in general, in transient growth inhibition of the i.m. transplants of lines X5, X6, and X7 provided that IL-2 was administered locally. The therapeutic effect depended on the number of transplanted tumour cells, the best results being achieved at cell numbers close to the dose-inducing tumour growth in 50% of animals. We found that the spontaneously arising tumours responding to IL-2 treatment were all slowly growing and immunogenic (X6 and X7) or might have viral association (X5) and, as such, might express foreign antigens. The data suggest a correlation between tumour immunogenicity and the therapeutic effect. However, IL-2 can still exert some effect against tumours with negligible immunogenicity. Received: 16 July 1998 / Accepted: 5 October 1998     

Host resistance to a spontaneously metastasising melanoma after primary tumour excision.

Tumour resistance was investigated in a spontaneously metastasising murine melanoma. Specific tumour resistance was found in mice with intact small primary tumours and no metastases. If primary tumours were excised, resistance was demonstrated in mice which had metastases and in mice which did not have metastases but were given crude melanoma homogenate. No resistance could be detected in mice which had large primary tumours left intact or in mice which had small primary tumours excised four days prior to tumour challenge. These in vivo findings are compared with previously documented in vitro results.     

Differential effects of nitrogen mustard in vivo on the cancer and host cells in MCa-11 tumours

We analysed the effects of nitrogen mustard (HN₂) on the growth, cell cycle distributions, and ratios of tumour cells to host cells for MCa-11 tumours grown in vivo. Treatment of tumour-bearing BALB/c mice with 3 mg/kg of HN₂ produced a significant slowing of MCa-11 tumour growth. Seventy-two hours after treatment in vivo with either 3 or 4 mg/kg of HN₂, the host cells in the treated tumours showed a significantly decreased G₀/G₁ peak and an increased G₂/M peak (P < 0.01), whereas the cancer cells in the treated tumours showed significant increases in the G₀/G₁ peak coupled with relatively decreased proportions of S and G₂/M tumour cells (P < 0.001). The ratio of the total number of cancer cells to the total number of host cells in the tumours was significantly increased 72 h after HN₂ administration (P<0.01). Thirty-two days after treatment with HN₂, the cell cycle distributions of the host and tumour cells in the treatment and control tumours had returned to being identical, but the ratio of the total number of cancer cells to the total number of host cells remained increased in the treated tumours (P<0.01). These results show that the administration in vivo of HN₂ can lead to entirely different cell cycle effects for the host and cancer cells in the same tumour, and that the partial growth arrest of MCa-11 tumours from HN₂ treatment may be due in part to the preferential destruction of host cells rather than solely to a direct cytotoxic effect on the cancer cells.