Treatment of hepatocellular carcinoma utilizing lymphokine-activated killer cells and interleukin-2.

This paper is a report on adoptive immunotherapy involving consecutive injections of recombinant interleukin-2 and lymphokine-activated killer (LAK) cells in the treatment of hepatocellular carcinoma. Peripheral blood lymphocytes, obtained by leukopheresis, acted as the activated killer cells with a co culture of recombinant interleukin-2 in the culture system. After 4 days, the activated killer cells were returned into the patients' bodies intra-arterially and intravenously. No complete remissions or partial remissions have resulted, although five of the seven patients showed a significant decrease in their serum alpha-fetoprotein levels after treatment. In addition, one case showed a patent portal truncus while another indicate the appearance of central necrosis on the computerized tomograph scan. Although the period of observation was short, there were no recurrences after the combination therapy of tumor resection and LAK adoptive immunotherapy. It might be difficult to treat hepatocellular carcinoma with adoptive immunotherapy alone, but there is some possibility of conducting therapy for hepatocellular carcinoma after removing the majority of the tumor cells by surgical resection and transcatheter arterial embolization therapy. This conclusion indicates, at least theoretically, that adoptive immunotherapy will be suitable in the treatment of hepatocellular carcinoma as one of the combination therapies with the two major forms of treatment mentioned above.

[Effects of various factors on the growth and function of a human hepatoblastoma cell line, HuH-6--an approach for serum-free cell culture].

The effects on a human hepatoblastoma cell line (HuH-6) of serum and Ca2+ were investigated. A higher concentration of serum reduced the production of alpha-fetoprotein, whereas Ca2+ enhanced that of both albumin and alpha-fetoprotein. In view of these facts, a serum-free medium using RPMI-1640 supplemented with lactalbumin hydrolysate, epidermal growth factor, hydrocortisone, insulin, chrelatoxine, glucagone and selenium was then developed. Collagen type IV was superior to collagen type I, laminin, fibronectin and plastic in supporting the growth of HuH-6 in the serum-free medium. Higher amounts of both albumin and alpha-fetoprotein were secreted in HuH-6 cultured in serum-free medium than in serum-supplemented medium.

[An attempt of in vitro portal invasion model of hepatocellular carcinoma utilizing permeable collagen membrane].

Hepatocellular carcinoma (HCC) possessed the ability of vascular invasiveness toward hepatic portal vein on the process of progression. This biological character of HCC can influence the patients survival on clinically. In this paper, we tried to establish the in vitro portal invasion model with human materials. The hepatic portal vein endothelial cell (HPVEC) derived from intrahepatic portal veins by surgically, have been propagated, as outgrowth cultures in RPMI-1640 medium with 10% fetal bovine serum, on permeable collagen membranes (KOKEN, Tokyo) containing mainly type I collagen, covered with a solubilized tissue basement membrane (MATRIGEL, Collaborate Res., Inc., Bedford MA) involving type IV collagen, laminin and proteoglycan. The primary cultured HPVEC with polygonal shaped cells forming a pavement stone sheet, were positively stained with Factor VIII related antigen and synthesized both prostacyclin and collagenase inhibitor. Co-culture of primary human HPVEC and HuH-7 (human HCC cell line obtained from Prof. Satoh, Okayama Univ.,) cells were inoculated onto reverse side between collagen membrane and gell formed basement membrane. Morphological alterations on the side of HPVEC can be obtained such as polylayered cells and different cytoplasmic cells among HPVEC. These results indicate that this experimental model can provide an useful in vitro model for the study of HCC portal invasion.

An attempt to eliminate fibroblast-like cells from primary cultures of fetal human livers.

The elimination of fibroblast-like cells from primary cultures of fetal human livers was studied. A fibroblast-like cell line (HuF), which was obtained by subculturing fetal human liver cells 4 or more times, was briefly treated with hydrocortisone (HC) or putrescine (PUT). The growth of HuF cells was inhibited by HC at a concentration of 10(-2) M and by PUT at a concentration higher than 10(-3) M. Long-term treatment of HuF cells with 10(-3) M HC inhibited the growth of the cells. Primary cultures of fetal human livers were made in medium containing HC or PUT, and morphological and functional examinations were made. The cultures were predominantly composed of epithelial-like cells, with few fibroblast-like cells, when the HC concentration was 10(-5)M to 10(-3) M. A high amount of albumin was secreted at these concentrations of HC. On the other hand, at 10(-3) M PUT, many epithelial-like cells were seen, but albumin was undetectable. The present results indicate that albumin-producing epithelial-like cells can be selectively maintained in medium containing HC, in primary cultures of fetal human livers.

Primary cultures of human livers and their albumin-producing capacity.

Primary cultures of surgically obtained noncancerous portions of human liver tissues were made. Liver tissues were poorly dissociated with collagenase, but well dissociated with dispase. The yield and viability of cells were improved somewhat when dissociated with collagenase followed by dispase. The mean cell yield was 1.1 X 10(6) cells/g liver. The epithelial-like morphology of the dissociated liver cells was maintained for about one week, but thereafter degenerative alteration of cells was observed. In liver explant culture, an active outgrowth of cells was observed for more than one month. Albumin production in culture fluids from dissociated livers was detectable for about 2 weeks, but later became undetectable, while that from explant culture was detectable for at least one month. These data demonstrate that adult human hepatocytes can be isolated from noncancerous portions of livers with relatively high yield, and that albumin production of the dissociated cells is detectable for several days.

Subcellular abnormalities of liver sinusoidal lesions in human hepatocellular carcinoma.

Liver sinusoidal lesions in 20 cases of human hepatocellular carcinoma (HCC) were examined with the electron microscope. Kupffer cells existed in the compact type of HCC. In area with pseudoglandular and trabecular cell patterns, Kupffer cells could not be observed. Only a few macrophages were distributed in the tumor tissues. Endothelial cells were altered and showed a poly-layered arrangement and were attached to each other with desmosome-like junctional complexes. There was a general loss of endothelial fenestrae. Endocytotic vesicles could be recognized in these endothelial cells. Poly-layered endothelial cells and accompanying layers of basal laminae were variably arranged between sinusoids and tumor cells with pseudoglandular, trabecular, or compact types of tumor cell patterns. Atypical cells containing numerous lysosomes, together with altered fat-storing (Ito) cells, were located in the tissue space bordering the capillaries. Moreover, tumor cells possessed flattened sinusoidal surfaces. These alterations of the sinusoidal wall suggest that capillarization of liver sinusoids in HCC took place, by loss of fenestrations, formation of basal laminae, and loss of microvilli on the surface of tumor cells. These architectural alterations are thought to completely change the physiological pattern of exchange of metabolites between tumor cells and the sinusoidal lumen. The absence of large numbers of Kupffer cells suggests that at this stage of tumor development, local cellular defense mechanisms were inoperative.

Production of basic fibroblast growth factor-like factor by cultured human cholangiocellular carcinoma cells.

An extract of cultured human cholangiocellular carcinoma cells (HuCC-T1) was found to contain high mitogenic activity for BALB/c3T3 cells. The growth factor eliciting most of the mitogenic activity was purified and concluded to be identical with basic fibroblast growth factor (bFGF)-like factor on the basis of its molecular weight and heparin-Sepharose elution profile, and the results of immunoblotting and radioimmunoassay. HuCC-T1 cells also secreted bFGF-like factor into serum-free medium. A combination of insulin and transferrin or bovine serum albumin stimulated the growth of HuCC-T1 cells in serum-free medium. However, bFGF did not stimulate their growth in the presence and absence of these supplements. Neutralizing monoclonal antibody against bFGF did not inhibit growth. These results indicate that bFGF-like factor is not a growth factor for this cell line.

Effects of various substrates on human hepatoblastoma and hepatoma cell culture.

The effects on human hepatoblastoma (HuH-6) and hepatoma (HuH-7) cell lines of collagen type I (CI), type IV (CIV), fibronectin (FN) and laminin (LAM) were investigated. CI and CIV promoted almost equally the attachment of both cell lines more strikingly than did FN or LAM. CI and CIV were also superior to FN or LAM in supporting the growth of HuH-6. These substrates, however, had no appreciable effect on the growth of HuH-7. The amount of alpha-fetoprotein (AFP) and albumin secreted in HuH-6 was found to be higher on FN- and LAM- coated substrates than on the other matrix material- coated ones. HuH-7 exhibited increased levels of AFP on LAM- coated substrates 4 days after plating. These results indicate that the ability of extracellular matrix materials to enhance attachment and/or growth is different from that to enhance AFP and albumin production in HuH-6 and probably in HuH-7.

A new human cholangiocellular carcinoma cell line (HuCC-T1) producing carbohydrate antigen 19/9 in serum-free medium.

A human cholangiocellular carcinoma cell line, HuCC-T1, was established in vitro from the malignant cells of ascites of a 56-yr-old patient. Histologic findings of the primary liver tumor revealed a moderately differentiated adenocarcinoma. Tumor cells from the ascites have been cultured with RPMI 1640 medium containing 0.2% lactalbumin hydrolysate and the cultured cells grew as monolayers with a population doubling time of 74 h during exponential growth at Passage 25. They had an epithelial-like morphology and were positive for mucine staining. Ultrastructural studies revealed the presence of microvilli on the cell surface and poorly developed organelles in the cytoplasm. The HuCC-T1 cell was tumorigenic in nude mice. The number of chromosomes in HuCC-T1 ranged from 61 to 80. These human cholangiocellular carcinoma cells in serum-free medium secreted several tumor markers, including carbohydrate antigen 19/9, carbohydrate antigen 125, carcinoembryonic antigen, and tissue polypeptide antigen. The carbohydrate antigen 19/9 secretion level of HuCC-T1 cells cultured in RPMI 1640 medium with 1% fetal bovine serum was sixfold higher than that with 0.2% lactalbumin hydrolysate. These findings suggest that HuCC-T1 will provide useful information to clarify the mechanism of tumor marker secretion and tumor cell growth in the human cholangiocellular carcinoma.

The effect of conditioned medium on fetal human liver cells in primary culture.

A medium conditioned by rat embryo cultures (RCM) promoted the adhesion of liver cells from human fetuses to plastic dish. Colonies were also formed in primary cultures of the same cells in the presence of RCM. The majority of the colonies formed were composed of large polygonal cells with a few colonies composed of both clear epithelial-like cells and fibroblast-like cells. RCM was superior to the rat embryo feeder layer for promotion of colony formation of cells. A number of colonies were formed from fetal human livers when a conditioned medium from human hepatoma cells (HCM) was used, but most of the colonies formed were composed of fibroblast-like cells. The cells derived from the polygonal cell colonies, which were formed in the presence of RCM, have been passaged four times and they are still growing with albumin-producing capacity. The effect of RCM was reduced by various physico-chemical treatments.

Thrombotic thrombocytopenia purpura caused by piperacillin successfully treated with plasma infusion.

An 81-year-old man was admitted to the hospital with a fever and loss of appetite. After treatment with piperacillin sodium (PIPC), the patient exhibited thrombocytopenia, hemorrhagic colitis, and drug-induced skin eruption. On the fifth day after PIPC induction, he further experienced neurological abnormalities, such as disorientation and confusion, renal dysfunction, and microangiopathic hemolytic anemia (MAHA). The patient was diagnosed with thrombotic thrombocytopenic purpura (TTP) on the basis of thrombocytopenia, MAHA, renal dysfunction, fever, and neurological abnormalities. Infusion of fresh-frozen plasma was initiated for treatment. His condition improved markedly after this treatment. It is rare for TTP to be accompanied with hemorrhagic colitis and skin eruption. These symptoms were induced by PIPC and were successfully treated with plasma infusion.

Induction of allogeneic tumour- and lymphokine-activated lymphocytes against hepatocellular carcinoma.

For clinical application of adoptive immunotherapy against hepatocellular carcinoma (HCC), it is not easy to prepare tumour specific effector cells such as cytotoxic T lymphocytes (CTL). To induce potent and broad-spectrum effectors, allogeneic cultured hepatoma cell lines (JHH-4 and HuH-6) were used as stimulators of peripheral blood lymphocytes (PBL) instead of autologous HCC cells. Allogeneic tumour- and lymphokine-activated killer cells (ATLAK) were generated by a mixed culture of lymphocytes and allogeneic cultured tumour cells with recombinant interleukin-2 (rIL-2). The tumour-killing activity of ATLAK induced by HuH-6 was confirmed against HuH-6 and other different HCC cell lines (JHH-2, HuH-7 and PLC). These activated lymphocytes were significantly more potent than lymphokine-activated killer cells (LAK) in [51Cr]-releasing assay. The JHH-4 stimulated ATLAK was reactive not only with JHH-4 but also with JHH-2. The lysis of allogeneic targets could be partially inhibited by anti-CD8 and anti-CD3 but not by anti-CD4. Anti-tumour cytotoxicity in these cultures might be mediated by CD3+CD56- and CD3+CD56+ effectors. These results imply that adoptive immunotherapy for HCC with ATLAK may be more feasible than that with LAK.