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Agarose microdroplet leukocyte migration inhibition (LMI) assays were performed to measure reactivity against line 10 hepatocarcinoma antigens and purified protein derivative (PPD) with the use of peripheral blood leukocytes from line 10 and/or BCG-sensitized syngeneic guinea pigs. The assay was quite sensitive and detected leukocyte migration inhibition with concentrations as low as 12.6 ng protein/ml of the crude sonicate of the line 10 tumor and 0.1 pg PPD. Specificity was shown by lack of reactivity in leukocytes of line 10 and/or BCG-sensitized animals with antigen preparations of L2C leukemia cells or normal syngeneic liver. Furthermore, leukocytes from normal control guinea pigs failed to react with any antigen. The results also suggested antigen cross-reactivity between line 10 tumor and BCG. Leukocytes from guinea pigs sensitized to only BCG became LMI reactive to the line 10 sonicate as well as PPD. No reactivity was observed with leukocytes of the animals in simultaneous tests with a sonicate of guinea pig L2C leukemia cells. The results demonstrated the usefulness of this microassay in detection of LMI reactivity with low antigen concentrations and small volumes of whole blood.

Direct leukocyte migration inhibition (LMI) assays were performed to investigate whether cell-mediated immune reactions could be detected in response to tumor-associated antigens of human melanoma. The antigens were 3 M KCl-soluble extracts of different fresh melanomas, other cancers, and benign nevus tissue. A total of 48 of the 79 (61%) blood samples from melanoma patients (64 patients) reacted with extracts of melanoma tissue. Since the subjects were usually tested with two or three extracts, 57/134 (42%) tests with melanoma patients' leukocytes were inhibited by KCl extracts of melanoma tissue, whereas only 3/50 (6%) tests with leukocytes of normal donors and 4/27 (15%) with patients having other cancers gave positive results. No positive reactions were obtained when 13 melanoma patients were tested with a 3 M KCl extract of benign nevus tissue. Likewise, only 2/26 (8%) positive tests were obtained from melanoma patients tested with extracts of other cancers. Individuals in all stages of disease had similar incidences of positive reactions to the soluble melanoma extracts, except for patients with stage-1 disease who exhibited a somewhat higher incidence of reactivity. The highest incidence of reactivity was observed in patients before surgical resection of the tumor, and somewhat decreased reactivity was seen 0-14 days post surgery. The results indicate that the direct LMI assay may be used to measure cell immune reactivity against melanoma-associated antigens. Since many of the positive results were obtained with allogeneic extracts, the results also indicate that different melanomas possess common antigens.

Macrophage migration inhibition assays, with a direct agarose microdroplet method, were used to monitor TAA activity of preparations of SV-40-induced mKSA cells. These preparations included cell-free crude membranes, papain-solubilized and NP40 detergent-solubilized membrane extracts from mKSA tumor cells. The assay was extremely sensitive and could detect migration inhibition reactivity with all three types of antigenic preparations with concentrations as low at 250 ng protein/ml. The reactivities were quite reproducible from experiment to experiment using the same or different lots of these antigen preparations, and the reactivities were specific in that peritoneal exudate cells from BALB/c mice, immunized with antigenically unrelated but syngeneic plasmacytomas, were not inhibited by these antigens. The results demonstrated the usefulness of this assay in rapidly detecting small concentrations of partially purified TAA preparations by using small number of immune cells.

Mononuclear (MN) cells from the peripheral blood of lung cancer patients were tested for their ability to respond to a 3-M KCl extract of adenocarcinoma of lung with the use of an indirect leukocyte migration inhibition (LMl) assay. Antigen-stimulated MN cell cultures were evaluated for leukocyte inhibitory factor production by their ability to inhibit the migration of indicator polymorphonuclear cells from agarose droplets. When supernatants were prepared in conventional round-bottomed tubes (5X10(6) cell/tube), 25 of 44 (57%) lung cancer patients had positive indirect LMl responses to the 7661 antigen as compared to only 2 of the 30 (7%) normal donors. When supernatants were prepared in conical microtubes, with 10 times fewer MN cells, similar results were obtained. Patients with all histologic types of lung cancer had a similar incidence of reactivity, and reactivity of untreated patients did not appear to be related to stage of disease or degree of tumor burden. Surgical removal of the tumor appeared to decrease the incidence of reactivity in the 1- to 12-month postoperative period. These results strongly suggest that the LMl reactivity against lung tumor extracts is lymphokine mediated, inducing cellular responses by the patients against such antigens.

BALB/c mice immunized with Nonidet P-40 (NP-40) crude solubilized (CS) extracts of a syngenetic methylcholanthrene-induced BALB/c sarcoma (Meth A) were challenged with viable Meth A cells to determine the ability of the solubilized preparations to induce transplantation rejection. Animals resisting such challenge were then used in agarose microdroplet macrophage migration inhibition (MMI) and tumor cell neutralization (Winn) assays to evaluate the antigenic specificity of these CS extracts. Spleen cells from those animals that rejected Meth A after immunization with the NP-40-solubilized preparations effectively neutralized the tumor-producing capacity of Meth A tumor cells as determined in Winn assays. MMI assays were quite sensitive and detected migration inhibition of peritoneal exudate (PE) cells from immunized mice with extract concentrations as low as picogram quantities. Specificity studies demonstrated that Meth A expressed no antigenic cross-reactivity with similarly prepared extracts of an unrelated SV40-induced sarcoma (mKSA), nor with a mineral oil-induced plasmacytoma (ADJ-PC5) of BALB/c mice. Inhibition of PE cell migration was mediated by culture supernatants (presumably migration inhibition factor [MIF]) generated from a mixture of immune spleen cells and mitomycin C (MMC)-treated Meth A cells as assayed in an indirect MMI test.

Cell-mediated immunity (CMI), as detected by the agarose microdroplet macrophage migration inhibition (MMI) assay, was investigated using peritoneal exudate cells (PEC) of BALB/c mice and several crude membrane (CM) and solubilized preparations of murine plasmacytomas. The MMI assay was quite sensitive and detected inhibition of macrophage migration as low as picogram quantities of CM, NP40 detergent- and papainsolubilized preparations (CS) of ADJ-PC5 and LPC-1 plasmacytomas. The data were highly reproducible from one experiment to the next with the same or different lots of the CM or solubilized extracts. Specificity studies demonstrated that ADJ-PC5 and LPC-1 plasmacytomas expressed cross-reactive tumor-associated antigens (TAA) as detected by MMI and confirmed by tumor challenge and Winn neutralization experiments. No cross-reactivity was observed with similar extracts prepared from an unrelated syngeneic simian virus 40 (SV40)-induced sarcoma. The inhibition of macrophage migration observed was mediated by culture supernatants generated from the mixture of plasmacytoma-immune spleen cells with antigens and then assayed in an indirect MMI assay on normal PEC. The agarose microdroplet MMI assay appeared to be a rapid and sensitive method to measure TAA recognition and to monitor TAA isolation and solubilization with minimum numbers of immune cells.

A high incidence of indirect leukocyte migration inhibition reactivity of normal donors to a 3-M KCl extract from a fresh pleural effusion of a patient with a lung adenocarcinoma (designated 7661) was observed. When these normal donors were classified according to contact with lung cancer patients or materials, 22 of 32 (72%) normal donors in contact with lung cancer patients or materials were reactive with the 7661 extract as compared to only 3 of 76 (4%) who had no contact. Of normal donors involved in the direct care of lung cancer patients, 14 of 20 (70%) were positive, whereas only 2 of 10 (20%) hospital personnel who worked with noncancer patients were reactive. Among laboratory personnel who handled blood and tissue specimens from lung cancer patients, 8 of 11 (73%) were positive with the 7661 extract, whereas none of 5 laboratory workers who worked with cancer materials unrelated to lung cancer were positive. Also, none of 13 personnel working in laboratories adjacent to those where lung cancer tests were performed were reactive with 7661. None of the 16 blood bank donors and none of 11 secretarial and clerical staff who worked in biochemical laboratories were positive. Reactivity was no correlated with a smoking history. Thus development of reactivity appeared to require direct contact with lung cancer patients or materials. The results suggested a horizontal transmission of reactivity against an antigen associated with lung cancer.

Direct leukocyte migration inhibition assays using the capillary tube technique can be used to demonstrate cell-mediated immunity in vitro. Unfortunately, the cumbersome nature of this technique makes it time consuming and difficult to perform. Similar results have been obtained using the direct agarose microdroplet leukocyte migration inhibition assay. In this paper, modifications of the agarose technique are outlined which insure standardization of droplets and ease of performance of the assay. Additionally a technique is described to reduce the time required for calculation of results.

The leukocyte migration inhibition assay was used to compare the antigenic reactivity of 3 M KCl extracts of human tumors. Many extracts demonstrated strong reactivity with patient leukocytes, whereas others demonstrated weak or no reactivity, Extracts prepared from primary tumors or local recurrent tumors were more antigenic than extracts from involved lymph nodes or pleural effusions. The least reactive preparations were extracts made from specimens of liver metastases obtained at autopsy. A large standard extract tested at a standard concentration was useful for the evaluation of antigenic reactivity of human tumor extracts. It served as a point of reference in simultaneous tests with one blood sample from each individual, thus eliminating the influence of patient variation on extract reactivity.

Studies were conducted to determine whether MCF-7, a tissue culture cell line derived from a pleural effusion of a patient with breast carcinoma, could be used as a source of tumor-associated antigen for direct leukocyte migration-inhibition (LMI) assays. Of 32 patients with breast carcinoma, 27 (84.4%) gave positive migration-inhibition results on their initial tests with a 25-mug protein/ml concentration of a 3 M KCl extract of MCF-7; 1 of 24 (4.5%) normal donors reacted with MCF-7. An intermediate incidence of reactivity (7/16) was observed with the extract when leukocytes of patients with melanoma, lung carcinoma, and Ewing's sarcoma were used. In further specificity studies, leukocytes of patients with breast carcinoma gave a lower incidence of LMl reactivity than did those of patients with Ewing's sarcoma and lung carcinoma with KCl extracts of the appropriate histologic type of tumor. The results indicated that the MCF-7 cells possessed a tumor-associated antigen to which many patients with breast carcinoma are sensitized.